SPECIAL METHODS. 185 



y. Saf ran in . 



320. The aniline-water-safranin recommended by Zwaar- 

 demaker (I) is best used for staining with this dye. It may 

 be prepared by mixing equal volumes of a concentrated 

 alcoholic solution of safranin and aniline-water * ; and 

 should be allowed to act for an hour or longer. Prepara- 

 tions may be washed with alcohol or acid alcohol, i.e., 

 alcohol containing about .5$ of hydrochloric acid. 



d. Gentian Violet (Gram's Method). 



321. The method originally proposed by Gram for staining 

 isolated bacteria (cf. 470) is in many cases well adapted for 

 staining nuclei, especially in material which has been fixed 

 with one of the acid mixtures recommended in 309, 312, 

 and 313. It is well to use here as a staining fluid, a mixture 

 of 3 grams of aniline, I gram of gentian violet, 15 grams of 

 alcohol, and 100 grams of water. The sections remain in 

 this one or a few minutes, the stain is washed off with 

 alcohol, and a solution of I part of iodine and 2 parts of 

 potassium iodide in 300 parts of water is at once added. 

 This gives the sections a dark color and is then washed off 

 with alcohol ; clove-oil is added, which extracts more color- 

 ing matter from the sections, and usually first brings out the 

 characteristic differential staining ; finally the sections are 

 mounted in Canada balsam. 



It may be especially remarked here that in this process 

 the action of the clove-oil is in no way merely a clearing 

 one, as has often been said. If it be replaced by xylol, even 

 after thorough washing with alcohol, not nearly so good 

 nuclear stains will be obtained, in many cases, as where clove- 

 oil is used. 



Finally, a very good double stain may be obtained by 

 dissolving eosin in the clove-oil used for washing. The walls 

 and the achromatic nuclear figure appear pure red, and the 

 -chromatic figure violet. 



* This is prepared by shaking water with an excess of aniline, and contains 

 about 3.5$ of aniline. 



