44 BACTERIOLOGY 



each ioo c.c. of the mixture. This final mixture is employed 

 in the same manner as Leishman's stain. It keeps for a few 

 months. 



Method of Staining Cover-glass Preparations. (a) A smear 

 preparation of bacteria having been made and fixed in the manner 

 above described, and a watery solution of either fuchsin, gentian 

 violet or methylene blue having been prepared, the cover-glass 

 is to be dropped into a dish containing the dye, or the dye may 

 be dropped upon the cover glass held in the forceps. 



(b) Allow the stain to act for about thirty seconds. 



(c) Wash in water. 



(d) Examine with the microscope in water directly or after 

 drying and mounting in Canada balsam. 



The rapidity and intensity of staining may be increased by 

 warming the solution slightly. The bacteria will usually appear 

 more distinct if, directly after pouring off the stain, the prepara- 

 tion is rinsed for a few seconds in i per cent solution of acetic 

 acid, and then thoroughly washed in water. The acetic acid 

 solution serves to remove in a measure any color which has 

 been imparted to the background, and which is undesirable. 



Preparations that are mounted at first in water may be made 

 permanent by moistening the edge of the cover-glass so that it 

 may be easily removed from the slide, then drying and mounting 

 in Canada balsam. Cover-glass preparations which have been 

 stained are examined with the oil-immersion objective, employ- 

 ing the plane mirror, having the iris diaphragm open and the 

 condenser close to the lower surface of the glass slide. The 

 purpose is to obtain the most intense illumination possible over 

 a small field. 



Gram's Method. Cover-glass preparations, having been 

 prepared and fixed in the usual manner (see page 38), are stained 

 as follows: 



(a) Stain in aniline-water gentian violet solution, from two 

 to five minutes. The intensity of the stain may be increased 

 by warming slightly. 



