Methods for Testing Stains 297 



is stained in a 1% aqueous crystal violet for 5 min., rinsed in distilled water and 

 transferred to equal parts of a 1% iodine and 1% KI in 80% alcohol for 15 to 50 

 seconds. The sections are next dehydrated in absolute alcohol for 4 to 5 seconds 

 and differentiated in a saturated solution of orange G in clove oil. They are then 

 cleared in xylene and mounted. In both of these procedures a stain is required 

 which gives a good contrast to the nuclear dye. 



Janus Green B,* C. I. No. 133 



Identification: Janus green B is diethylsafranin-azo-dimethylanilin, CsoHaiNeCl. 

 It is identified by the following method: Dissolve 50 mg. in 125 ml. of 95% alcohol 

 and then dilute to 250 ml. with distilled water. Take 10 ml. of this solution and 

 dilute to 200 ml. with 50% alcohol. Read in Beckman spectrophotometer. Absorp- 

 tion maximum 610-628 m/x; ratio P-15/P+15 is from 0.98 to 1.02. 



Method of analysis: Dissolve 100 mg. of dye in 50 ml. of 95% alcohol, add 150 ml. 

 of distilled water and 10 g. of sodium acid tartrate, heat to boiling, and titrate with 

 0.05 N TiCla. 



The dye solution undergoes several color changes on reduction, but the final end 

 point, usually a clear reddish-brown, is easy to recognize. The following data are 

 used to calculate the percentage of anhydrous dye in the original sample: 



Molecular weight 511 .053 



Hydrogen equivalents per mol of dye 6. 



ml. of N/10 TiCla per gram of dye 117.405 



Samples of this stain should contain not less than 50% anhydrous dye. 



Biological Tests: Janus green is tested, mixed with neutral red, for the supravital 

 staining of blood. Saturated stock solutions of the two dyes are prepared by adding 

 125 mg. of neutral red to 50 ml. of neutral absolute alcohol and 125 mg. of Janus 

 green to 62.5 ml. of neutral absolute alcohol. Both of these solutions are stable. A 

 dilute stock solution of neutral red is prepared by adding 1.1 ml. of the saturated 

 neutral red to 10 ml. of neutral absolute alcohol. This solution is also stable. In 

 testing Janus green, 0.4 ml. of the saturated solution of this dye is added to 

 3 ml. of the dilute solution of neutral red. This mixture is not stable and must be 

 used promptly. 



Slides are washed and dried carefully, after treatment in bichromate cleaning 

 fluid, and are then flooded with the mixed stain, which is allowed to drain off. The 

 slides are then dried quickly in front of a Bunsen flame. A drop of fresh blood is 

 placed on a cover slip which is inverted on top of the slide having the film of dried 

 stain. The edges of the cover slip must be sealed with vaseline of high melting point. 



By this technic, with good samples of both dyes, the basophilic granules become 

 brilliant scarlet, the eosinophilic granules yellow or light orange, while the neu- 

 trophilic granules are salmon color. It is essential that no staining of the nuclei 

 should be obtained which, if it occurs, indicates that the cells are killed. All the 



*The formula given in the Colour Index for Janus green B has been found incor- 

 rect for all samples furnished in this country. The dye usually encountered as a 

 biological stain has the formula given here. However, German samples of the dye, 

 although similar, are not identical with the usual stain sample. 



