Methods for Testing Stains 323 



Erythrosin B, C. I. No. 773 



Idetiiification: Erythrosin is the sodium salt of tetraiodofluorescein, CsoHeOal*- 

 Na2. Identification is by the following method: Dissolve 50 mg. in 250 ml. of dis- 

 tilled water. Dilute 5 ml. of this solution and 2 ml. of 1% NazCOs solution to 200 

 ml. with distilled water. Read in Beckmau spectrophotometer. Absorption maxi- 

 mum 524-526.5 m/x; ratio P-15/P+15 is from 1.15 to 1.41. 



Method of Analysis: Dissolve an accurately weighed sample of about 0.5 g. of the 

 dye to be examined in approximately 500 ml. of water, heat to boiling and add 

 slowly with constant stirring, 2 or 3 ml. of 6 N hydrochloric acid. Cool the hot solu- 

 tion to room temperature and allow to stand at least 1 or 2 hours. Filter the pre- 

 cipitated color acid on a weighed Gooch crucible, thoroughly wash with a 0.2% 

 solution of hydrochloric acid, dry at 110° C, and weigh. Calculate the dye content 

 of the original sample from the weight of the color acid by means of the following 

 formula : 



Wt. of Color Acid X 879.922 X 100 , , , , 



835.944 X Wt. of Original Sample = P^^ ^^^^ ^^ anhydrous dye. 



Note: 879.922 = Molecular weight of erythrosin. 



835.944 = Molecular weight of the color acid of erythrosin. 



Samples of this stain should not contain less than 80% anhydrous dye. 



Biological Tests: Erythrosin is tested in the Jackson stain (1926) for plant 

 anatomy. In employing this procedure as a test for erythrosin, plant buds or other 

 botanical material containing both lignified and non-lignified cell walls are fixed in 

 one of the usual fixatives, embedded in paraflSn and sectioned. After removing the 

 paraflan they are rinsed with absolute alcohol, then 95% alcohol, and stained 

 15-30 minutes in 1% aqueous crystal violet. Then, after quickly rinsing in distilled 

 water and dehydrating, they are differentiated 1-5 minutes in a saturated solution 

 of erythrosin in clove oil, cleared in xylene-alcohol, 1:1, passed through xylene and 

 mounted. By this method the non-lignified tissues should stain with erythrosin and 

 the lignified walls with crystal violet. 



Phloxine B. C. I. No. 778 



Identification: Phloxine B is the sodium salt of tetrabromotetrachlorofluorescein, 

 C2oH205Cl4Br4Na2. Identification is as follows: Dissolve 50 mg. in 250 ml. of 50% 

 alcohol. Dilute 5 ml. of this solution and 2 ml. of 1% Na2C03 solution to 200 ml. 

 with 50% alcohol. Read in Beckman spectrophotometer. Absorption maximum 

 547-548 mfjL-, ratio P-15/P+15 is from 1.38 to 1.62. 



Method of Analysis: Precipitate the color acid of a weighed sample of the dye to be 

 tested by the method outlined for the analysis of erythrosin B. From the weight of 

 the color acid, the dye content of the original sample is calculated by means of the 

 following formula: 



Wt. of Color Acid X 829.702 X 100 . , , j 



Wt. of the Original Sample X 785.724 = P^^ ^^^^ ^^ anhydrous dye. 



Note: 829.702 = Molecular weight of phloxine B. 



785.724 = Molecular weight of the color acid of phloxine B. 



Samples of this stain should not contain less than 80% anhydrous dye. 



