38 



86. The time of staining. Although in general certain time 

 limits can be given to the period during which a stain should be 

 allowed to act, with most stains, especially those with which no dif- 

 ferentiation is needed, such as hematoxylin, and most carmines, the 

 correct intensity of color should be determined by examining the 

 preparation with the microscope. One soon becomes able to judge 

 of the right stain in this way better than if a given time were adhered 

 to. 



In the use of stains requiring a subsequent differentiation, the 

 rule is 'to over-stain and watch the differentiation carefully with the 

 microscope, stopping it when sufficient. In this case it is the differ- 

 entiation and not the staining that should be carefully regulated. 

 In general, for the best results, it is advisable to use staining and 

 differentiating solutions in dilute form and prolong the time during 

 which they act. 



The following formulas include the more generally useful stains 

 and those to be employed in the "Special Methods" given subse- 

 quently. 



STAINS. 



87. Hematoxylin Stains. Hematoxylin is a colorless compound of acid 

 properties forming therefore salts with bases which oxidize readily forming 

 "hemateates." The oxidation product of hematoxylin is hematein which is 

 the real staining principle and may in some cases be used with real advantage 

 instead of hematoxylin. Hematoxylin (hematein) itself has little value as an 

 acid (plasma) stain; combined with metallic bases it becomes a valuable basic 

 (chromatin) stain. The metals usually employed as mordants for hematoxylin 

 are: aluminium, iron, copper, chromium, molybdenum, vanadium. Their 

 salts may be used either in the same solution as the hematoxylin (aluminium, 

 molybdenum, vanadium) or separately (iron, copper.) Solutions of the same 

 metals may in some instances (aluminium, iron, chromium,) be employed also as 

 differentiator. For preparing hematoxylin stains it is a great convenience to 

 have a 10% stock solution in 95% alcohol. 



88. Chloral Hematoxylin. [11] Formula: Potassium alum, 8 grams; 

 distilled water, 250 c. c.; hematoxylin 2/10th gram or 2 c. c. hematoxylin stock 

 solution. Boil 5 or 10 minutes in an agate dish. After cooling add 6 grams of 

 chloral hydrate. Place in a bottle and permit the hematoxylin to oxidize for a 

 week or two, or 1 to 2 c. c. hydrogen peroxid may be added. Its staining quality 

 improves up to an optimum and then begins to deteriorate. Old hematoxylin 

 generally contains a precipitate and should be filtered often or before using. 



Stain sections 5 to 30 minutes according to the age of the solution, the charac- 

 ter of the tissue and the fixation employed. After staining wash well with dis- 

 tilled or tap water. Usually no differentiation is required unless a purer chroma- 

 tin stain is desired ( 175). Counter stain as desired. 



