296 Biological Stains 



or (2) Molecular weight, (Ci 0H6N2O5) 2Ca 506.392 



Hydrogen equivalents per mol of dye 24. 



ml. of N/10 TiCla per gram of dye 473.944 



Biological tests: Martins yellow is tested as a counterstain to resorcin blue for 

 staining pollen tubes in the style. Slender styles and ovaries, while still moist, are 

 crushed between two slides; while larger ones are treated similarly after sectioning 

 longitudinally by hand. The material is either stained on the slide or immersed 

 in the stain in pieces in a small dish, for 2 to 5 minutes. The staining solution con- 

 sists of 5 mg. resorcin blue and 5 mg. martins yellow in 10 to 15 ml. water, with a 

 few drops of 1% aqueous ammonia added to bring the reaction to about pH 8, as 

 shown by the solution assuming an olive color. The material is mounted in the 

 stain or else in water of the same reaction, and examined with a powerful light. A 

 good sample is one with which the pollen tubes show blue on a light yellowish green 

 background. 



Okange G, C. I. No. 27 



Identification: Orange G is the disodium salt of benzene-azo-2-naphthol-6:8-disul- 

 fonic acid, Ci6HioN207S2Na2. It is identified by the following method: Dissolve 

 50 mg. of orange G in 250 ml. of distilled water. Dilute 15 ml. of this solution to 

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

 maximum 476-481 m/z; ratio P-15/P+15 is from 0.88 to 0.94. 



Method of Analysis: Dissolve 10 mg. of dye in 175 ml. of distilled water, add 

 25 ml. of acetate bujffer pH 4.0, heat to boiling, and titrate with 0.05 A^ TiCl|. 

 The end point is a sharp change from brownish-yellow to a pale yellowish-green. 

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

 original sample: 



Molecular weight 452.370 



Hydrogen equivalents per mol of dye 4. 



ml. of N/10 TiCla per gram of dye 88.424 



For certification, samples of this stain must contain not less than 80% anhydrous 

 dye. 



Biological Tests: Orange G is tested (1) in Mallory's connective tissue stain and 

 (2) as a counterstain in histology and (3) in cytology. 



It is tested in Mallory's connective tissue stain on animal tissue fixed in Zenker's 

 solution and embedded in paraffin. The sections are stained 1-5 minutes in a 0.5% 

 aqueous solution of acid fuchsin and transferred directly to a solution containing 

 0.5 g. anilin blue W.S., 2 g. orange G, and 100 ml. of a 1% aqueous solution of 

 phosphotungstic acid, for 10 to 20 minutes or longer; dehydrated in several changes 

 of 95% alcohol, then absolute alcohol; cleared in xylene and mounted in xylene, 

 colophonium or balsam. With a good sample, the red blood corpuscles and myelin 

 sheaths are yellow and elastic fibers pale pink or yellow. 



For its histological use it is tested on animal tissue fixed in Bouin's or Zenker's 

 solution and embedded in paraffin. A 0.5% solution in 95% alcohol is employed and 

 applied for 15 seconds or more as a counterstain after Heidenhain's hematoxylin. 

 (See Eosin Y). 



For cytological work, root tip material, fixed in Navashin's or Flemming's fluid, 



