Methods for Testing Stains 311 



white precipitate is obtained upon addition of dilute AgNOs, the original dye con- 

 tained cliloride. 



Method of Analysis: Dissolve 200 mg. of dye in 120 ml. of distilled water, add 

 50 ml. alcohol, 10 ml. of glacial acetic acid and 30 ml. of 30% sodium tartrate 

 solution, heat to boiling and titrate slowly with 0.05 N TiCls to a light straw color. 

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

 sample: 



(1) Molecular weight, C23H25N2CI 364.903 



Hydrogen equivalents per mol of dye 2. 



ml. of N/10 TiCls per gram of dye 54.810 



(2) Molecular weight, 2C23H25N2 + 3C2H2O4 928.000 



Hydrogen equivalents per mol of dye 4. 



ml. of N/10 TiCls per gram of dye 43.057 



Samples of this stain should contain not less than 75% anhydrous dye (if the 

 chloride) or not less than 90% dye (if the oxalate). 



Biological Tests: Malachite green is tested for use as a counterstain in plant his- 

 tology. Botanical material (preferably plant pathological material), after fixation 

 in a suitable fixative (e. g., Flemming's, Carnoy's or Farmer's fluid) and embedding 

 in paraflBn, is sectioned and stained with 0.5% malachite green in 95% alcohol, 

 distilled water, or in clove oil, either with or without previous staining in 1% 

 aqueous safranin. The malachite green is applied for one minute when used alone, 

 or for 20 seconds following 20 minutes of the safranin. When used alone, the stain 

 should reveal clearly all such histological elements as the cell walls, endodermis, 

 bast, cytoplasm, nuclei, and chloroplasts. When used following safranin on plant 

 pathological material, the host nuclei, xylem, and cutinized walls, as well as the 

 nuclei of the infecting fungus, should appear red. The cytoplasm and cellulose walls 

 of the host should appear green. 



It is also tested in the SchaefiFer and Fulton spore stain for bacteria and in Conk- 

 lin's modification of the method. For the former, bacterial smears are fixed in a 

 flame and flooded with 5% aqueous malachite green for 30-60 seconds, then 

 heated to steaming three or four times. They are washed in water 30 seconds 

 and 0.5% aqueous safranin is added for 30 seconds; they are then washed and 

 blotted dry. The spores should be green, the rest of the cells red. 



For the latter method, bacterial smears are fixed in a flame and flooded with 5% 

 aqueous malachite green and allowed to steam 10 min. The slides are washed in 

 running water 30 seconds and counterstained 1 minute with 5% aqueous mer- 

 curochrome. The spores should be green, the rest of the cells red. 



BRILLLA.NT Green, C. I. No. 662 



Identification: Brilliant green is the acid sulfate of p, p -tetraethyldiaminotri- 

 phenylcarbinol anhydride, C27H33N2SO4H. The method of identification is as fol- 

 lows: Dissolve 50 mg. in 250 ml. of 50% alcohol. Dilute 3 ml. of this solution to 

 200 ml. with 50% alcohol. Read in Beckman spectrophotometer. Absorption 

 maximum 628-€31 m/i; ratio P-15/P+15 is from 0.94 to 1.08. 



