STAIN TECHNIQUE 11 



continues until the section is washed with distilled water. After a 

 second rinse, in distilled water containing a trace of ammonium 

 hydroxide, the oxidation of Hematein is completed and the object 

 is stained deep blue-black. If the material is washed with tap 

 water containing sulfuric acid, the pigment is greenish black. 



Oxidation. In microtechnique, the study of the conditions 

 which effect color is especially important ; the behavior of biological 

 stains differs with the physical and chemical properties of the 

 tissues and with the presence of chemical assistants. Many coal- 

 tar colors are more fast if they are developed by means of oxida- 

 tion. The importance of this reaction can be demonstrated with 

 a number of azo dyes which do not stain plant tissues unless an 

 oxidizing agent is added to the dye bath. If Erythrosin, a Xan- 

 thene dye, is absorbed by a tissue in the presence of potassium di- 

 chromate, the pink is changed to scarlet. With many of the coal- 

 tar dyes, however, the oxidized color is soluble in water or alcohol ; 

 consequently such stains are unsuited for permanent mounts. 



In aqueous solutions of Picric Acid, plant and animal tissues 

 remain pale yellow. With the addition of acetic acid to the stain, 

 the yellow is increased but fades after five hours exposure to sun- 

 light. With the aid of potassium dichromate and acetic acid, the 

 color changes very little after more than 50 hours exposure to sun- 

 light. Thus, the presence of appropriate chemical assistants strik- 

 ingly modifies the fastness of Picric Acid. 



Reduction. Dyes may be affected by reduction in a number 

 of ways. In a glycerin mount, ferric ferrocyanide fades to white 

 ferrous ferrocyanide unless a trace of potassium dichromate is 

 added to the dilute glycerin during dehydration. Most of the sul- 

 fur, mordant, and vat dyes insoluble in water or alcohol are 

 brought into solution by reduction. After the tissue has retained 

 the stain in its soluble state, the color is developed by re-oxidation. 

 The oxygen necessary to complete the action may be absorbed from 

 the air. • Hydrochloric acid in acid-alcohol changes many basic dyes 

 to the leuco compound, the form of dye which is not necessarily 

 colorless but which is usually more fugitive. For this reason, the 



