Methods for Testing Stains 301 



Method of Analysis: Dissolve 0.1 g. of the dye in 125 ml. of distilled water, add 30 

 ml. of 30% sodium tartrate solution, heat to boiling, and titrate with N/10 TiCls. 

 Titrate rapidly until near the end point, then slowly. The end point is sharp, a 

 practically colorless solution resulting. Data for the calculation of the percentage 

 of anhydrous dye in the sample are as follows: 



Molecular weight 696.658 



Hydrogen equivalents per mol of dye 8. 



ml. of N/10 TiCls per gram of dye 114.834 



For certification, samples of this stain must contain not less than 75% anyhdrous 

 dye. 



Biological Tests: Congo red is tested on paraflBn sections of animal tissue fixed in 

 Bouin's fluid. The sections are stained 5 minutes in Mayer's hemalum, dipped 

 in tap water once or twice, and transferred directly into 0.5% aqueous solution of 

 Congo red and left therein for 1 minute. They are then rinsed in tap water, run up 

 through the alcohols, cleared and mounted. The criteria by which the sample is 

 judged are as follows: Congo red should give a bright cytoplasmic stain with a cer- 

 tain amount of differentiation from orange to reddish. Erythrocytes, for instance, 

 should be light orange and the spindle fibers in mitotic figures should be deep orange 

 to light red. 



Chlorazol Black E, C. I. No. 581 



Identification: Chlorazol black E has the empirical formula C34H25N907S2Na2. It 

 is identified as follows: Dissolve 50 mg. in 250 ml. of 50% alcohol. Dilute 5 ml. of 

 this solution to 200 ml. with 50% alcohol. Read in Beckman spectrophotometer. 

 Absorption maximum 598-602 m/i; ratio P-15/P+15 is from 0.97 to 1.00. 



Method of Analysis: No method of analysis has been devised for chlorazol black E. 



Biological Tests: Chlorazol black E is tested on paraffin sections of animal tissue 

 fixed in Zenker's fluid, and on plant tissue fixed in Flemming's or Bouin's. The 

 sections are stained 5-10 minutes in a 1% solution in 70% ethyl alcohol. The excess 

 dye is drained off and the sections are dehydrated, cleared and mounted in balsam. 

 No mordant and no differentiation are necessary. The tissue elements should stain 

 varying shades of green, gray and black, and sharp differentiation should be evi- 

 dent. Too general appearance of green is regarded as undesirable. 



In using this as an auxiliary stain in making chromosome counts on plants, the 

 above staining solution is applied 5-25 minutes to the dissected tissue, fixed in acetic 

 acid (1 volume to 3 volumes of alcohol), rinsed in three changes of 70% alcohol 

 and the material is then transferred to a slide. A drop of aceto-carmine (boiling 

 45% acetic acid saturated with carmine and filtered) is added and then covered 

 with a cover glass, heated, flattened and sealed. The chromosomes should stain deep 

 reddish black with a fairly clear cytoplasm. 



Alizarin Red S, C. I. No. 1034 



Identification: Alizarin red S has the empirical formula C14H7O7S Na. It is 

 identified as follows: Dissolve 50 mg. in 250 ml. of 0.1 N NaOH. Dilute 15 ml. of 

 this solution to 200 ml. with 0.1 N NaOH. Read in Beckman spectrophotometer. 



