EXAMINATION AND STAINING OF BACTERIA 49 



mixed and steamed in the sterilizer for one hour. When cold add 

 0.1 per cent aqueous solution of eosin in the proportion of 5 parts 

 eosin solution to 6 parts methylene blue solution. The mixture 

 becomes a purple color and a granular sediment appears. It is 

 then filtered and the precipitate remaining on the filter paper is 

 pressed dry. A saturated solution is made of the dried precipitate 

 in methyl alcohol; this saturated solution is then filtered and 

 diluted by the addition of 10 c.c. of methyl alcohol to 40 c.c. of 

 the stain. 



In using the stain a few drops are placed on a fixed film for 

 one minute, then the same quantity of water is dropped on to 

 the slide by means of a medicine dropper and the mixture of stain 

 and water is allowed to remain two to three minutes. The slide 

 is then washed in water and dried. 



The stain gives particularly good results in the examination 

 of blood films. Erythrocytes appear yellow or pink; the nuclei 

 of leucocytes various shades of purple and the cytoplasm a light 

 blue color; blood plaques dark blue; bacteria blue. Malarial 

 parasites stain characteristically : the chromatin mass appears 

 a garnet red and the surrounding protoplasm a robin's egg blue. 



Gram's Stain. The fixed film is covered with a fresh solution 

 of aniline gentian violet made as follows : One c.c. of anilin oil is 

 added to 10 c.c. of water and shaken until thoroughly emulsified, 

 after which it is filtered through wet filter paper. One part of 

 saturated alcoholic gentian violet is then added to nine parts of 

 the filtrate. The slide is allowed to remain in the above dye for 

 five minutes, after which it is immersed in the following iodine 

 solution for 2 to 3 minutes. 



Iodine 1 gm. 



Potassium iodide 2 gm. 



Distilled water 300 c.c. 



The film is then decolorized with 97 per cent alcohol about one 

 minute or until no more stain can be washed out of the preparation, 

 after which it is washed in water and counterstained with eosin 

 for thirty seconds. 

 This method of staining is frequently used in bacterial differen- 



