298 Biological Stains 



areas that stain with neutral red take up the dye quickly, except in the case of 

 monocytes, which require about 10 minutes. The mitochondria show up after 

 about 15 minutes as small blue dots or rods. The cells should live in good condition 

 for at least an hour. 



Methyl Orange, C. I. No. 142 



Identification: Methyl orange is the sodium salt of p-sulfobenzene-azo-dimethyl- 

 anilin, CuHuNsOsSNa. It is identified by the following method: Dissolve 50 mg. 

 in 250 ml. of distilled water. Dilute 5 ml. of this solution and 1 ml. of lA^ HCl to 

 200 ml. with distilled water. Read in Beckman spectrophotometer. Absorption 

 maximum 506-508 m^i, ratio P-15/P+15 is from 0.95 to 0.96. 



Method of analysis: Dissolve 100 mg. of dye in 200 ml. of distilled water, add 

 15 g. sodium acid tartrate, heat to boiling, and titrate with 0.05 N TiCls. The end 

 point is sharp, giving a colorless solution. The following data are used in calculating 

 the percentage of anhydrous dye in the original sample: 



Molecular weight 327.333 



Hydrogen equivalents per mol of dye 4. 



ml. of N/10 TiCla required per gram of dye 122.200 



Samples of this stain should contain not less than 85% anhydrous dye. 



Biological Tests: Methyl orange is tested in saturated alcoholic solution as a 

 counterstain, applied for 15 minutes following crystal violet, or 1 or 2 minutes fol- 

 lowing Harris' or Heidenhain's hematoxylin, on paraffin sections of animal tissue 

 fixed in Bouin's or of plant tissue fixed in Flemming's fluid. A good sample is one 

 which shows a good contrast to the nuclear stain. 



Orange II, C. I. No. 151 



Identification: Orange II is the sodium salt of p-sulfobenzene-azo-2-naphthol, 

 Ci6HiiN204SNa. It is identified by the following method: 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 483-487 m/x; ratio 

 P-15/P4-15 is 0.96 



Method of analysis: Dissolve 100 mg. of dye in 200 ml. of distilled water, add 10 g. 

 of sodium acid tartrate, heat to boiling and titrate with 0.05 N TiCls. Reduce to a 

 practically colorless solution. The following data are used in calculating the per- 

 centage of anhydrous dye in the original sample: 



Molecular weight 350.321 



Hydrogen equivalents per mol of dye 4. 



ml. of N/10 TiCls per gram of dye 114.179 



Samples of this stain should contain not less than 85% anhydrous dye. 



Biological Tests: Orange II is tested by the same procedure as in the case of 

 methyl orange. 



