Methods for Testing Stains 309 



throcytes of Necturus are utilized as test materials. With Paramecium aqueous so- 

 lutions ranging from 1:5000 to 1:100,000 concentration are employed, and one drop 

 of concentrated culture is added to 10 ml. of the dye solution. The rate and in- 

 tensity of staining of intracellular granules, the effect on ciliary activity and ele- 

 vation of the pellicle, and the distortion of the organism constitute the criteria of 

 the suitability of the dye for vital staining. In supravital testing a 1:1250 solution 

 in absolute ethyl alcohol is applied to a glass slide, yielding the so-called dry-dye- 

 film. Drops of freshly drawn blood are placed on the slide, covered, and sealed 

 with warm vaseUne. Observations are made at room temperature with the prepa- 

 ration shielded from direct heat from the source of illumination. In the erythrocyte, 

 definite bipolar clusters of preformed granules stain immediately. Relative toxicity 

 is judged by the rate at which induced or secondary staining patterns (granules or 

 reticulation) appear. Corrections for percentage of dye content and the kind of 

 salt (chloride or iodide) are necessary. 



In testing for the second purpose (staining living blood and connective tissue 

 cells), the same procedure is used as for Janus green, see p. 297. 



Safbanin O, C. I. No. 841 



Identification: Safranin is a mixture of diaminophenylditolazonium chloride, 

 C20H19N4CI and diamino-o-tolylditolazonium chloride, C21H21N4CI. The method of 

 identification is: 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. Ab- 

 sorption maximum 530-533 mn; ratio P— 15/P+15 is from 1.14 to 1.32. 



Method of Analysis: Spectrophotometric. Percent dye = D-peak X 231. D-peak 

 (color density at peak) measured on dye solution described under "Identification". 



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

 dye. 



Biological Tests: Safranin is tested as a chromosome stain in the Flemming triple 

 stain and in combination with light green SF yellowish or fast green FCF, also the 

 Gram stain. The procedures for the Flemming stain are the same as described 

 below under crystal violet (p. 313). The procedure with fast green or light green as 

 a stain is given on page 313, 



NiGROsiN, Water Soluble, C. I. No. 865 



Identification: Nigrosin water soluble is the sodium salt of the product resulting 

 from the sulfonation of spirit soluble nigrosin which is obtained by the interaction 

 of anilin, anilin hydrochloride and nitrophenol (or nitrobenzene and iron). The 

 method of identification is: Dissolve 50 mg. in 250 ml. of 50% alcohol. Dilute 25 ml. 

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

 Absorption maximima 575 m/x', ratio P-15/P-1-15 is from 0.94 to 0.97. 



Method of Analysis: Since this dye is a variable mixture of complex compounds of 

 unknown constitution, quantitative determination of the dye content is impossible. 



