306 Biological Stains 



prepared as the chloride. It is identified as follows: Dissolve 50 mg. in 250 ml. of 

 distilled water. Dilute to 5 ml. of this solution to 200 ml. with distilled water. 

 Read in Beckman spectrophotometer. Absorption maxim mn 620-632.5 nxfji; ratio 

 P-15/P+15 is from 1.01 to 1.09. 



Method of Analysis: Dissolve 100 mg. of dye in 175 ml. of water, add 25 ml, of 

 pH 4.0 acetate buffer (6 volumes of 50% Na Ac -31120 + 4 volmnes glacial acetic 

 acid), heat to boiling and titrate with 0.05 N TiCla to a yellow endpoint. The 

 following data are used in calculating the percentage of anhydrous dye in the 

 sample : 



Molecular weight 305.819 



Hydrogen equivalents per mol of dye 2. 



ml. of N/10 TiCls per gram of dye 65.398 



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

 dye. 



Biological Tests: Toluidine blue O is tested as a stain for frozen sections and as a 

 substitute for azure A in Haynes' modification of French's stain. For the former 

 purpose the same procedure is used as in the case of thionin, p. 303; for the latter the 

 same as in the case of azure A, p. 304. 



It is also tested as a constituent of Albert's diphtheria stain, using the following 

 method: Smears are made' and fixed with gentle heat. They are stained 5 minutes 

 in the following solution: toluidine blue, 0.15 g.; methyl green, 0.02 g.; glacial acetic 

 acid, 1 ml.; 95% ethyl alcohol, 2 ml.; and distilled water, 100 ml. The stain is 

 drained off without washing, and Lugol's iodine solution is applied for 1 minute; 

 the smears are then washed briefly in tap water, blotted with filter paper and ex- 

 amined. A good stain shows the granules of the diphtheria bacilli black, the bars 

 of bacilli dark green to black, the body of the cells and other bacteria light green. 

 It is also tested in Laybourn's modification in which malachite green is used in- 

 stead of methyl green. 



Brilliant Cresyl Blue, C. I. No. 877 



Identification: In general, samples of this stain are aminodimethylaminomethyldi - 

 phenazonium chlorides, CieHieNaOCl, although the dye is given in the Colour 

 Index as the diethyl derivative. The method of identification is: Dissolve 50 mg. 

 in 125 ml. of distilled water and then dilute to 250 ml. with 95% alcohol. Dilute 

 5 ml. of this solution to 200 ml. with 50% alcohol. Read in Beckman spectrophoto« 

 meter. Absorption maximum 626-630 m/x; ratio P-15/P-|-15 is from 1.10 to 1.21. 



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

 ml. of distilled water and 10 g. of sodiinn acid tartrate, heat to boiling and titrate 

 with 0.05iV TiCls to the first appearance of a lighter reddish-brown color. The 

 following data are used in calculating the precentage of anhydrous dye in the 

 original sample: 



Molecular weight 289.759 



Hydrogen equivalents per mol of dye 2. 



ml. of N/10 TiCls per gram of dye 69.023 



