Methods for Testing Stains 305 



It is also tested as a constituent of the tetrachrome stain made up as follows: 

 methylene blue chloride, 1.0 g.; azure A, 0.6 g.; methylene violet, 0.2 g.; eosin Y, 

 1.0 g. are mixed together; 0.15-0.3 g. of the mixed dry ingredients are dissolved in 

 100 ml. methyl alcohol (neutral, acetone free) by heating to 50°C., shaken thoroughly 

 and left 1-2 days at 37°C., with occasional shaking. It is then filtered, after which it 

 is tested by the same procedure as Wright's stain, page 327. With a good azure A 

 this compound stain should color the platelets, the lobes of the polymorphonuclear 

 cells and the nuclei of the lymphocytes a distinct purple. 



AZUBE C 



Identification: Azure C is monomethyldiaminodiphenazthionium chloride (mono- 

 methyl thionin), C13H12N3SCI. The method of identification ia as follows: Dissolve 

 50 mg. in 250 ml. of distilled water. Dilute 3 ml. of this solution to 200 ml. with 

 distilled water. Read in Beckman spectrophotometer. Absorption maximum 

 607.5-612.5 mix; ratio P-15/P-(-15 is from 0.98 to 1.03. 



Method of analysis: Dissolve 100 mg. of dye in 175 ml. of distilled water, add 

 25 ml. of pH 4.0 acetate buffer (6 volumes of 50% Na Ac "31120 + 4 volumes of 

 ■glacial acetic acid), heat to boiling and titrate with 0.05 N TiCls to a yellow end- 

 point. The following data are used in calculating the percentage of anhydrous dye 

 in the sample: 



Molecular weight 277.767 



Hydrogen equivalents per mol of dye 2. 



ml. of N/10 TiCls per gram of dye 72.003 



Because of the uncertain composition of samples of this stain, in that they con- 

 tain varying amounts of the higher methylated thionins and thionin, no dye content 

 is reported. 



Biological Tests: Azure C is tested in the same way as azure A (see methylene 

 azure, p. 304.) 



Methylene Violet 



Identification: Dissolve 25 mg. in 125 ml. of 95% alcohol, add 10 ml. of O.liV HCl" 

 and dilute to 250 ml. with distilled water. Dilute 10 ml. of this solution and 20 ml. 

 of 0.1 N HCl to 200 ml. with 50% alcohol. Read in Beckman spectrophotometer. 

 Absorption maximum 579-580 m/z; ratio P-15/P+15 is from 0.99 to 1.02. 



Method of Analysis: Spectrophotometric. Percent dye = D-peak X 181. D-peak 

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



Biological Tests: It is tested as a constituent of the tetrachrome stain made up by 

 the formula given on p. 245. A blood smear stained with this mixture, containing 

 a satisfactory methylene violet, shows a pure blue in the cytoplasm of the lympho- 

 cytes and a deeper blue in the granules of this cytoplasm. 



ToLuiDiNE Blue O, C. I. No. 925 



Identification: Toluidine blue is usually the zinc chloride double salt of aminodi- 

 methylaminotoluphenazthionium chloride, CisHieNsSCl -|- ZnCU, but may also be 



