326 Biological Stains 



uncertain; the molecular formula is C22H20O13. It is a strong dibasic acid with a 

 side chain which contains four hydroxyl groups and possibly possesses a sugar-like 

 structure, although carminic acid is not a glucoside. It contains at least one asym- 

 metric carbon. 



Method of Analysis: Due to the uncertainty of its constitution, no dye content is 

 determined for samples of this stain. 



Biological Tests: In testing carmine, Mayer's alcoholic hydrochloric acid stain is 

 prepared by dissolving 4 g. carmine by boiling in 15 ml. distilled water with 30 

 drops of concentrated hydrochloric acid, then adding 95 ml. of 85% alcohol and 

 neutralizing by adding ammonia until the carmine begins to precipitate. This is 

 employed for staining chick or pig embryos fixed in Bouin's or Zenker's fluid. For 

 sections, the embryos are stained in bulk 24 to 72 hours, without decolorization, 

 embedded in paraffin, and sections are cut 10 to 15 n thick. For toto mounts of 

 both chick and pig embryos, the stain is applied for about 24 hours to secure deep 

 staining, decolorized until rose-pink in 1% HCI in 80% ethyl alcohol, dehydrated 

 and cleared. Small embryos are mounted in balsam and larger ones in benzyl ben- 

 zoate. A good sample of carmine is indicated by the degree of nuclear staining 

 secured. 



Carmine is also tested in Schneider's aceto-carmine formula (saturated solution 

 in boiling 45% glacial acetic acid) for staining chromosomes in smears of anthers. 

 The anthers are squeezed out gently onto the slide. A small drop of carmine is 

 quickly put on the anthers, and a cover slip is placed on it. It is heated gently and 

 then examined. The result should be a dark translucent red stain, selective for 

 chromatin, with un colored cytoplasm. 



Hematoxylin 



Identification: Hematoxylin is a logwood product whose exact chemistry is not 

 entirely understood, although its formula is assumed to be as given on p. 202. Be- 

 cause of this imcertainty, reliable chemical methods for its identification and analy- 

 sis have not yet been devised. 



Biological Tests: It is tested on paraflin sections of rabbit embryo by Delafield's, 

 Heidenhain's, and Ehrlich's technics, as follows: 



Delafield's hematoxylin is made up as follows: hematoxylifa, 4 g. is dissolved in 

 25 ml. 95% ethyl alcohol and 400 ml. saturated aqueous A1NH4(S04)2-12H20 is 

 added; this is allowed to stand a week exposed to air and light; it is then filtered 

 and 100 ml. glycerin and 100 ml. methyl alcohol is added. This solution becomes 

 dark in 6-8 weeks after which it is tested. Sections are stained for 15 minutes in 

 this solution after diluting with equal parts of water; they are then rinsed in tap 

 water and immersed in fresh tap water for about 10 minutes. If the sections are still 

 very blue, 2 drops of acidulated 35% alcohol (containing 3 drops cone. HCI to 50 

 ml.) are added to them on the slides and they are returned to the water. They are 

 then passed through 95% alcohol and absolute alcohol into xylene and are mounted 

 in balsam. A satisfactory sample should show blue nuclei. 



Heidenhain's technic is as follows: The paraffin is removed from the sections in 

 the usual manner. The sections are mordanted in 1.5-4% aqueous NH4Fe(S04)2- 

 I2H2O for 30 minutes to 3 hours, rinsed in tap water, stained 1-3 hours in 0.5% 



