Methods for Testing Stains 307 



For certification, samples of this stain must contain not less than 50% anhydrous 

 dye. 



Biological Tests: In testing brilliant cresyl blue, fresh blood is examined under u 

 cover glass on which a filtered 0.3% alcoholic solution of brilliant cresyl blue has 

 been dried or blood films are made on similar cover glasses, dried and counterstained 

 with Wright's or similar stain. The reticulum of immature red cells should be a 

 clear-cut blue on a very pale blue (fresh) or eosin colored (stained) background. 

 Blood platelets should stain a pale blue and be discrete, with only a minimum of 

 precipitate or debris. 



Nile Blue Sulfate (Nile Blue A), C. I. No. 913 



Identification: Nile blue A is aminodiethylaminonaphthophenazoniura sulfate 

 (C2oH2oN30)2S04. The method of identification is: Dissolve 50 mg. in 125 ml. of 

 95% alcohol and then dilute to 250 ml. with distilled water. Dilute 3 ml. of this 

 solution to 200 ml. with 50% alcohol. Read in Beckman spectrophotometer. Ab- 

 sorption maximum 635-645 m^i, ratio P-15/P-|-15 is from 1.02 to 1.12. 



Method of Analysis: Dissolve 100 mg. of dye in 100 ml. of 95% alcohol, add 75 

 ml. of distilled water and 25 ml. of 30% sodium tartrate solution, heat to boiling 

 and titrate with 0.05 N TiCla. It is necessary to keep the solution boiling throughout 

 the reduction, otherwise the reaction is very slow, especially near the end point. 

 The final color change is from reddish brown to yellow. The following data are used 

 in calculating the percentage of anhydrous dye in the original sample: 



Molecular weight 732.820 



Hydrogen equivalents per mol of dye 4. 



ml. of N/10 TiCls per gram of dye 54.584 



Samples of this stain should contain not less than 70% anhydrous dye. 



Biological Tests: Nile blue sulfate is tested as a dififerential fat stain by the Smith 

 and Mair technic. The material used for this test consists of insect ova fixed and 

 preserved in neutral formalin. Sections are cut on the freezing microtome and 

 stained with the hydrolyzed dye prepared by the technic given in the following 

 paragraph. After staining for about 2 hours at 37° C. they are dififerentiated in 2% 

 acetic acid, washed in distilled water and mounted in glycerin or Farrant's medium. 

 A good sample is indicated by its ability to distinguish between free fatty acids 

 (blue) and neutralized fats (red). The staining solution is prepared as follows: 



A saturated aqueous solution of Nile blue sulfate is made and 0.5% H2SO4 is 

 added. It is boiled under a reflux condenser for 1-2 hours. The solution is tested by 

 shaking up a little of it in a test-tube with xylene; if a suflScient amount of the 

 red oxazone has been formed, the xylene will assume an intensely fluorescent red 

 color. The H2SO4 may be neutralized by adding an equivalent amount of NaOH 

 or the stain may be used in acid solution. 



Cresyl Violet 



Identification: The American stain, cresyl violet (National Aniline Company) is 

 aminonaphthodimethylaminotolazoxonium chloride, CigHisNsOCl. On the other 



