108 BIOLOGY AND TECHNIQUE 



however, can be given here. In principle, all these stains depend upon a 

 combination of eosin and methylene-blue, these elements staining not 

 only as units, but acting together in combination. One and the same 

 solution, therefore, contains at least three elements which color the 

 various structures of the preparation selectively. 



JENNER'S METHOD. 1 This stain, because of its simplicity, is useful 

 for routine use. It is made up as follows: Equal parts of eosin (Gruebler, 

 "W. G.") one and two-tenths per cent aqueous solution, and methylene- 

 blue (medicinal, Gruebler) one per cent aqueous solution, are mixed and 

 allowed to stand for twenty-four hours. A coarse granular precipitate 

 is formed which appears dark, with a metallic luster on its surface. This 

 is separated by filtration and washed with distilled water until the ni- 

 trate appears almost clear. 



To make up the stain 0.5 gram of the dry precipitate is dissolved 

 in 100 c.c. of methyl alcohol. 



In using the stain, preparations are not fixed, but simply dried in 

 the air and immersed in the stain for one to two minutes. After this, 

 wash in distilled water and examine. 



WRIGHT'S MODIFICATION OF LEISHMAN'S METHOD. 2 A one per cent 

 solution of methylene-blue (Gruebler) in five-tenths per cent solution of 

 sodium bicarbonate in distilled water is steamed in a sterilizer at 100 

 C. for one hour. After this has cooled, a one-tenth per cent aqueous 

 solution of eosin (Gruebler, W. G.) is added until a metallic scum ap- 

 pears on the surface of the mixture. (About five parts of eosin solution 

 to one of methylene-blue is necessary.) The precipitate which forms is 

 collected by filtration, dried, and a saturated solution then made in 

 methyl alcohol. This is filtered and diluted with one-quarter its bulk 

 of methyl alcohol. 



To stain, cover the dried preparation with the stain for one to 

 one and one-half minutes. Dilute by dropping upon the stain distilled 

 water from a pipette until a metallic film appears upon the top. Leave 

 this on for three to fifteen minutes. Wash in distilled water. 



GIEMSA'S METHOD. 3 The method of Giemsa is really a modification 

 of the Romanowsky method. It is widely applicable, being of great 

 value in the staining of the Spirochsete pallida, Vincent's spirilla, pro- 

 tozoa, and Negri bodies. The stain has been modified several times by 



1 Jenner, Lancet, i, 1889. 



2 Wright, Jour. Med. Research, ii, 1902. 

 * Giemsa, Cent. f. Bakt., I, xxxvii, 1904. 



