46 THE MICROSCOPIC PREPARATION. 



Hematoxylinusafranin of Rabl (85). Sections of preparations 

 fixed with chromic-formic acid or with a solution of platinum chlorid are 

 stained for a short time with Delafield's hematoxylin, then counterstained 

 for twelve to twenty-four hours with safranin and washed with absolute 

 alcohol until no more color is given off. 



Biondi-Heidenhain Triple Stain. Of the many triple stains in 

 use we mention only the most important, the rubin S orange G 

 methyl-green mixture of Ehrlich and Biondi, employed according to the 

 modification of M. Heidenhain. The best results are obtained with ob- 

 jects fixed in saline sublimate solution. The three stains just mentioned 

 are prepared in concentrated aqueous solutions. (In 100 c.c. of distilled 

 water there are dissolved respectively about 20 gm. of rubin S, and 8 

 gm. of orange G and methyl-green.) These concentrated solutions are 

 combined in the following proportions: rubin S 4, orange G 7, methyl- 

 green 8. The stock solution thus obtained is diluted with 50 to 100 

 times its volume of distilled water before using. The sections should be 

 as thin as possible and fixed to the slide by the water method. They 

 remain for twenty-four hours in the stain, and are then rinsed in distilled 

 water or in 90% alcohol or in such with the addition of a little acetic acid 

 (i to 2 drops to 50 c.c. ). Before staining it is occasionally of advantage 

 to treat the sections with acetic acid (2 : 1000) for one to two hours. 



STAINING IN 'BULK. 



Instead of staining in sections, entire objects can be stained before 

 cutting. This method is in general much slower, and demands, there- 

 fore, special staining solutions, as, for instance : 



Alcoholic Borax-carmin Solution. Pieces ^ cm. in diameter remain 

 in the stain at least twenty-four hours, are then decolorized for the same 

 length of time in acid alcohol (0.5% to \ c / c hydrochloric acid in 70% 

 alcohol), and after washing in 70% alcohol are transferred to 90^ alco- 

 hol. Larger objects require a correspondingly longer time. 



Parficarmin. Treatment as in section staining; length of time 

 according to size of object. 



Alum-carmin of Grenacher. This never overstains. Time of stain- 

 ing according to size of object. Wash in water, then transfer to 70% 

 and 90^ alcohol. 



Hemalum, when diluted with water, is very useful for staining in bulk. 

 After staining, objects should be washed with distilled water. 



Bohmer's hematoxylin stains small pieces very sharply. Use the same 

 as hemalum. 



Hematoxylin staining according to R. Heidenhain's method is 

 especially recommended for staining in bulk. 



Stain objects fixed in alcohol or picric acid twenty-four hours in a 

 -33% aqueous solution of hematoxylin ; transfer for an equal length of 

 time to a 0.5% aqueous solution of potassium chromate, changing often 

 until the color ceases to run. Wash with water and pass into strong 

 alcohol. This stain also colors the protoplasm, and is so powerful that 

 very thin sections are an absolute condition to the clearness of the prepa- 

 ration . 



If the objects have been fixed with picric acid and the latter has 

 not been entirely washed out, staining in bulk by the above methods pro- 

 duces very striking differentiation. 



