Methods for Testing Stains 319 



tain only the disodium trisulfonate. Samples of acid fuchsin are considered for 

 analytical purposes as a mixture of equal parts of trisulfonated rosanilin and para- 

 rosanilin, unless specific information concerning the character of the fuchsin used 

 for the starting material has been obtained from the dye manufacturer. Identifica- 

 tion is by the following method: Dissolve 50 mg. of sample in 250 ml. of distilled 

 water. Dilute 6 ml. of this solution and 5 ml. of 0.1 N HCl to 200 ml. with distilled 

 water. Read in Beckman spectrophotometer. Absorption maximum for pararo- 

 sanilin 540-542 mju, for rosanilin 543-546 m^u; ratio P-15/P+15 for pararosanilin 

 is from 1.13 to 1.26, for rosanilin from 1.11 to 1.15. 



Andrade Indicator: Acid fuchsin is tested as to its behavior in the Andrade indi- 

 cator as follows: A 0.2% solution of the dye in 100 ml. of distilled water is decolor- 

 ized by adding normal NaOH a little at a time. Complete decolorization should be 

 brought about without the use of more than 25 ml. of the sodium hydroxide. (The 

 quantity of NaOH required should be stated on the label.) 



Method of Analysis: Spectrophotometric. Percent dye (for pararosanilin) = 

 D-peak X 142. Percent dye (for rosanilin) = D-peak X 151. D-peak (color den- 

 sity at peak) measured on dye solution described under "Identification". 



For certification, samples of this stain must contain not less than 55% anhydrous 

 dye. 



Biological Tests: Acid fuchsin is tested in the Van Gieson connective tissue stain 

 on animal tissue fixed in formalin or other suitable fixing fluid and embedded in 

 paraflBn. The paraffin is removed in the usual manner; the sections are stained 

 deeply in alum-hematoxylin or Weigert's iron-hematoxylin, washed in distilled water 

 and stained 3-5 minutes in Van Gieson's solution, (5 ml. of 1% aqueous acid fuchsin 

 and 100 ml. of saturated aqueous picric acid). They are then quickly washed in tap 

 water, differentiated in 95% alcohol, dehydrated, cleared and mounted in balsam. 

 The tissue elements stained by the acid fuchsin should show a clear red, contrasting 

 well with hematoxylin and picric acid. A good sample does not show great tendency 

 to fade. 



Acid fuchsin is also tested in the anilin blue connective tissue stain on animal 

 tissue fixed in Zenker's fluid and embedded in paraffin. The paraffin is removed in 

 the usual manner and the sections are stained in a 0.5% aqueous acid fuchsin for 

 1-5 minutes or longer, depending on the freshness of the tissue. The sections are 

 transferred directly to the following solution (anilin blue W.S., 0.5 g.; orange G, 

 2.0 g.; 1% aqueous phosphotungstic acid, 100 ml.) for 10 minutes or longer, dehy- 

 drated in several changes of 95% alcohol, then absolute alcohol, cleared in xylene 

 and mounted in balsam. The same criteria are used in judging a good sample with 

 this technic as with the Van Gieson stain. 



Anilin Blue, Water Soluble, C. I. No. 707 



Identification: Anilin blue consists of mixtures of the various sulfonation products , 

 of variable mixtures of phenylated rosanilin and pararosanilin, usually the latter. 

 The method of identification is as follows: Dissolve 50 mg. in 250 ml. of distilled 

 water. Dilute 10 ml. of this solution and 10 ml. of 0.1 iV HCl to 200 ml. with distilled 

 water. Read in Beckman spectrophotometer. Absorption maximum 595-610 m/x; 

 ratio P-15/P+15 is from 0.98 to 1.09. 



