44 THE MICROSCOPIC PREPARATION. 



tilled water 90 c.c. This solution should remain in an open vessel for 

 about four weeks, and, before using, should be diluted with an equal 

 volume of distilled water. Staining takes place in twelve to twenty-four 

 hours, after which the sections are rinsed in tap -water and again placed 

 in a like solution of ammonium sulphate of iron, until black clouds cease 

 to be given off from the sections. They are rinsed in distilled water, 

 passed through alcohol into xylol, and mounted in balsam. Should a 

 protoplasmic stain be desired, rubin in weak acid solution may be 

 employed. 



Coal=tar or anilin stains. Ehrlich classifies all anilin stains as salts 

 having basic or acid properties. The basic anilin stains, such as safra- 

 nin, methylene-blue, methyl-green, gentian violet, methyl-violet, Bis- 

 marck brown, thionin, and toluidin-blue are nuclear stains, while the 

 acid anilin stains, such as eosin, erythrosin, benzopurpurin, acid fuchsin, 

 lichtgriin, aurantia, orange G, and nigrosin stain diffusely and are used as 

 protoplasmic stains. 

 Safranin : 



Safranin , . . I gm. 



Absolute alcohol 10 c.c. 



Anilin water . . 90 " 



Anilin water is prepared by shaking up 5 c.c. to 8 c.c. of anilin oil in 100 c.c. of 

 distilled water and filtering through a wet filter. Dissolve the safranin in the anilin 

 water and add the alcohol. Filter before using. 



Stain sections of tissues fixed in Flemming's or Hermann's solutions 

 for twenty-four hours, and decolorize with a weak solution of hydrochloric 

 acid in absolute alcohol (i : 1000). After a varying period of time (usu- 

 ally only a few minutes) all the tissue elements will be found to have 

 become bleached, only the chromatin of the nucleus retaining the color. 



Bismarck Brown. A very convenient color to handle is 

 Bismarck brown. Of this, i gm. is boiled in 100 c.c. of water, filtered, 

 and */$ of its volume of absolute alcohol added. Bismarck brown stains 

 quickly without overstaining, and is also a purely nuclear stain. Wash 

 in absolute alcohol. 



Methyl-green stains very quickly (minutes), i gm. is dis- 

 solved in 100 c.c. of distilled water to which 25 c.c. of absolute alcohol 

 is added. Rinse sections in water, then place for a few minutes in 70% 

 alcohol, transfer to absolute alcohol for a minute, etc. 



Other so-called basic anilin stains can be used in a similar 

 manner. Thionin or toluidin-blue in dilute aqueous solutions are espe- 

 cially useful. Nuclei appear blue and mucus red. 



Double Staining. When certain stains are used in mixtures 

 or in succession, all portions of the section are not stained alike, but 

 certain elements take up one stain, others another. This elective affin- 

 ity of tissues is taken advantage of in plural staining. If two stains are 

 employed, one speaks of double staining. 



Picrocarmin of Ranvier. Two solutions are prepared, a satu- 

 rated aqueous solution of picric acid and a solution of carmin in ammonia. 

 The second is added to the first to the point of saturation. The whole is 

 evaporated to one-fifth of its volume and filtered after cooling. The 

 solution thus obtained is again evaporated until the picrocarmin remains 

 in the form of a powder. A i % solution of the latter in distilled water 

 is the fluid used for staining. 



