300 Biological Stains 



Sudan IV solution (Sudan IV, 0.1 g. in a mixture of 50 ml. 70% alcohol and 50 ml. 

 acetone C.P.), They are washed quickly in 70% alcohol and transferred to distilled 

 water, and counterstained in alum hematoxylin, washed thoroughly in tap water 

 and mounted in glycerin or glycerin jelly. A good sample should give a sharp 

 reddish-orange color to the fat globules. 



Bismarck Brown Y, C. I. No. 331 



Identification: Bismarck brown Y is the dihydrochloride of benzene-m-disazo-bis- 

 m-phenylenediamine, C18H20N8CI2. It is identified as follows: Dissolve 50 mg. in 

 125 ml. of 95% alcohol, then dilute to 250 ml. with distilled water. Dilute 10 ml. of 

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

 Absorption maximum 461-475 m/*; ratio P-15/P-|-15 is 1.01. 



Method of Analysis: Dissolve 100 mg. of the dye to be tested in 100 ml. of 95% 

 alcohol and 100 ml. of distilled water, add about 10 g. of sodium acid tartrate, heat 

 to boiling, and titrate with 0.05A'^ TiCls. The end point, which ranges from pale yel- 

 low to brownish-yellow, is not always sharp, and is best observed by adding the 

 titanous chloride solution 4 drops at a time. The following data are used for cal- 

 culating the percentage of anhydrous dye in the original sample: 



Molecular weight 419.318 



Hydrogen equivalents per mol of dye 8. 



ml. of N/10 TiCls per gram of dye 190.787 



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

 dye. 



Biological Tests: Bismarck brown Y is tested in a 1% aqueous solution for stain- 

 ing mucous in goblet cells of the intestine, or cartilage in the trachea or in embryonic 

 material, fixed in one of the usual fixatives and embedded in paraffin. The sections 

 are stained for about 5 to 10 minutes, rinsed in 95% alcohol, and transferred to the 

 0.5% aqueous methyl green solution until they appear dark green, after which they 

 are dehydrated, cleared and mounted. A good Bismarck brown should show light 

 brown mucous and deep brown cartilage. 



It is also tested in Neisser's method for diphtheria. Smears are prepared in the 

 usual manner and fixed with gentle heat. They are stained 2 or 3 seconds in acetic 

 methylene blue (methylene blue, 0.1 g.; 95% alcohol, 2 ml.; glacial acetic acid, 

 5 ml.; distilled water, 95 ml.). They are then washed in tap water; stained 3 to 5 

 seconds in 0.2% Bismarck brown dissolved in boiling water, which has been filtered; 

 washed, dried and unmounted. A good stain will show the bacilli uniformly brown 

 or may show at one or both ends, dark blue round body. True diphtheria organisms 

 usually show blue bodies, pseudotypes few. 



Congo Red, C. I. No. 370 



Identification: Congo red is the disodium salt of diphenyl-disazo-bis-l-naphthyl- 

 amine-4-sulfonic acid, C32H22N606S2Na2. It is identified as follows: Dissolve 50 mg. 

 of Congo red in 250 ml. of distilled water. Dilute 10 ml. of this solution and 2 ml. 

 1% Na2C03 to 200 ml. with distilled water. Read in Beckman spectrophotometer. 

 Absorption maximum 497-499 m/x; ratio P-15/P-1-15 is from 1.00 to 1.03. 



