324 Biological Stains 



Biological Tests: Phloxine B is tested by the Mallory phloxine and methylene blue 

 technic, by the procedure given in Staining Procedures, Leaflet lA, using 5% 

 phloxine instead of 2% eosin B. A satisfactory stain is judged by the pink color 

 of the cytoplasm, while the nuclei are colored by the methylene blue. 



Rose Bengal, C. I. No. 779 



Identification: Rose bengal is the sodium salt of tetraiodotetrachlorofluorescein, 

 C2oH205l4Cl4Na2. The following method of identification is employed: Dissolve 

 50 mg. in 250 ml. of distilled water. Dilute 5 ml. of this solution and 2 ml. of 1% 

 Na2C03 solution to 200 ml. with distilled water. Read in Beckman spectrophotom- 

 eter. Absorption maximum 544-550 mju; ratio P-15/P-|-15 is from 1,21 to 1.63. 



Method of Analysis: The method for the determination of rose bengal is identical 

 with that of erythrosin B, except that in the calculation of the dye content of the 

 original sample, the following formula is to be used in place of the one given for the 

 after dye. 



Wt. of Color Acid X 1017.718 X 100 ^ , 



Wt. of Original Sample X 973.740 = P^*" ^^"^ °^ anhydrous dye. 



Note: 1017.718 = Molecular weight of rose bengal. 



973.740 = Molecular weight of the color acid of rose bengal. 



For certification, samples of this stain should contain not less than 80% anhy- 

 drous dye. 



Biological Tests: Rose bengal is tested in Conn's technic for staining bacterik in 

 soil. The procedure is to stain a dried drop of soil suspension made by mixing soil 

 with 5-10 times its weight of 0.015% gelatin in distilled water. While on a flat 

 surface over boiling water this film is stained 1 minute with 1% rose Bengal in 5% 

 aqueous phenol, containing 0.01%, more or less, of CaCb. The amount of CaCl2 is 

 varied if unsatisfactory results are obtained with 0.01%, on the theory that in- 

 creasing its concentration intensifies the action of the stain. A sample is judged 

 satisfactory which permits a deep staining of the bacteria without sufficient coloring 

 of the soil or dead organic matter to obscure the microorganisms. In making the 

 test, soil known to have an unusually large number of bacteria is employed. 



MISCELLANEOUS DYES 

 Pyronin Y, C. I. No. 739 



Identification: Pyronin Y is tetramethyldiaminoxanthenyl chloride, C17H19N2- 

 OCl. Identification is by the following method: Dissolve 50 mg. in 250 ml. of 50% 

 alcohol. Dilute 5 ml. of this solution to 250 ml. with 50% alcohol. Read in Beckman 

 spectrophotometer. Absorption maximum 547-550 m/j.; ratio P-15/P-[-15 is 1.40. 



Method of Analysis: Spectrophotometric. Percent dye — (D-peak X density) 

 -H 1.58. D-peak (color density at peak) measured on dye solution described under 

 "Identification". Note: 1.58 is equal to the value of Holmes and Peterson (1930). 



For certification, samples of this stain must contain not less than 45% anhydrous 

 dye. 



