Methods for Testing Stains 325 



Biological Tests: Pyronin is tested in staining gonorrhoea! smears by the Pappen- 

 heim-Saathof stain, as given under methyl green, p. 318. With a good sample, the 

 cocci should be bright red and the nuclei green (not purplish). 



Pyronin B, C. I. No. 741 



Identification: Pyronin B is tetraethyldiaminoxanthenyl chloride, C21H27N2OCI. 

 Identification is by the following method: Dissolve 50 mg. in 250 ml. of 50% alcohol. 

 Dilute 5 ml. of this solution to 200 ml. with 50% alcohol. Read in Beckman spec- 

 trophotometer. Absorption maximum 554e-555 m/i; ratio P-15/P+15 is from 1.49 

 to 1.61. 



D-peak X 100. 

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



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

 Samples of this stain should contain not less than 30% anhydrous dye. 

 Biological Tests: Pyronin B is tested by the same procedure as pyronin Y. 



Indigo Carmine (Indigotine), C. I. No. 1180 



Identification: Indigo carmine is the disodium salt of indigotin-5,5 -disulfonic 

 acid, Ci6H8N208S2Na2. The following method of identification is used: Dissolve 

 50 mg. in 250 ml. of distilled water. Dilute 10 ml. of this solution to 200 ml. with 

 distilled water. Read in Beckman spectrophotometer. Absorption maximum 

 608-611 m^t; ratio P-15/P+15 is 1.04. 



Method of Analysis: Dissolve 200 mg. of dye in 200 ml. of distilled water, add 15 g 

 of sodium acid tartrate, heat to boiling and titrate with 0.05 N TiCla solution. The 

 color change at the end point is from blue to reddish brown. The following data 

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



Molecular weight 466.354 



Hydrogen equivalents per mol of dye 2. 



ml. of N/10 TiCls per gram of dye 42.895 



Samples of this stain should contain not less than 80% anhydrous dye. 



Biological Tests: Indigo carmine is tested in Shumways' modification (1926) of the 

 Cajal stain. Young amphibian larvae fixed in Bouin's picro-formol-acetic are em- 

 bedded in paraflSn and sectioned. Sections are stained 20 minutes in basic fuchsin 

 (sat. aq. sol.) after which they are rinsed in distilled water and placed in an equal 

 mixture of the indigo carmine to be tested and picric acid (each in sat. aq. sol.) for 

 5 minutes. They are passed rapidly through 70% alcohol (when the sections appear 

 red), 95% and absolute alcohol (until the sections appear green) and cleared in 

 xylene. With a satisfactory sample muscle tissue is green and connective tissue blue, 

 contrasting sharply with the red of the nuclei. 



Carmine, C. I. No. 1239 



Identification: Carmine is a cochineal product, usually furnished as the aluminum- 

 calcium salt of carminic acid. The exact structure for carminic acid is somewhat 



