302 Biological Stains 



This dye has two absorption maxima, one at 555-558 m/x, the other at 594-599 

 m/i, ratio P— 15/P+15 for first maximmn is 1.02. 



Method of Analysis: No method of analysis for alizarin red S is known. 



Biological Tests: Alizarin red S is tested on small vertebrates, fixed 2-4 days or 

 longer in 95% alcohol, by the following technic: Place specimen in 1% aqueous 

 KOH until the bones are clearly visible through the surrounding tissues, transfer 

 to dilute (0.0025-0.01%) alizarin red S in 1% aqueous KOH and allow to stand 

 until desired degree of staining is obtained; complete the clearing by placing in a 

 mixture of 1 vol. glycerin to 4 vol. of 1.25% aqueous KOH (glycerin 20 ml., KOH 

 1 g., water 79 ml.) and continue through increasing concentrations of glycerin. 

 Store in glycerin alone. The bones should be red, soft tissue transparent and un- 

 stained. 



Sudan Black B 



Identification: The empirical formula for Sudan black B is C24H22N6. The method 

 of identification is as follows: Dissolve 50 mg. in 250 ml. of 95% alcohol. Dilute 

 10 ml. of this solution to 200 ml. with 95% alcohol. Read in Beckman spectro- 

 photometer. Absorption maximum 596-605 m^i; ratio P-15/P+15 is from 0.99 to 

 1.02. 



Method of Analysis: A method for determinating dye content has not yet been 

 perfected. 



Biological Tests: Sudan black B is tested for fatty material in bacteria in Burdon's 

 technic. Culture of B. cereus is grown at room temperature for 18-24 hr. on potato 

 slant or glucose starch agar. Prepare film and air dry. Flood entire slide with 

 Sudan black B solution (Sudan black B 0.3 g., 70% alcohol, 100 ml.). After bulk of 

 dye has been dissolved, shake thoroughly at intervals during the day and allow to 

 stand over night. Flood slide with the stain and allow it to remain undisturbed 

 5-15 min., drain off excess stain, blot dry, clear with xylene, blot dry. A satis- 

 factory sample should show intercellular fat dark blue, cytoplasmic membrane 

 intensely stained, dark blue black. 



THE QUINONE-IMINE DYES 



Thionin (Lauth's Violet), C. I. No. 920 



Identification: Thionin is diaminodiphenazthionium chloride, C12H10N3SCI. It is 

 identified by the following method: Dissolve 50 mg. in 250 ml. of distilled water. 

 Dilute 5 ml. of this solution to 200 ml. with distilled water. Read in Beckman 

 spectrophotometer. Absorption maximum 598-599 mfx; ratio P— 15/P+15 is from 

 1.15 to 1.35. 



Method of Analysis: The determination of dye content is made by the spectre- 

 photometric method, calculating by the formula: Percent dye = D-peak X 97. 

 D-peak (color density at peak) is measured on dye solution as described under 

 'Identification". 



Samples of this stain should contain not less than 85% anhydrous dye. 



