THE MICROSCOPE AND MICROSCOPIC METHODS 45 



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 shde. The 

 purpose is to obtain the most intense illumination possible over 

 a small field. 



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

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

 as follows: 



(a) Stain in carbol gentian violet or in aniline-water gentian 

 violet solution, from two to five minutes. The intensity of the 

 stain may be increased by warming slightly. 



(b) Gram's solution, one and one-half minutes : 



. Iodine i gram. 



Potassium iodide 2 grams. 



Water 300 c.c. 



In this solution the preparation becomes nearly black. 



(c) Wash in alcohol repeatedly; the alcohol becomes stained 

 with clouds of violet coloring matter; the alcohol is used as long 

 as the violet color continues to come away, and until the prepara- 

 tion is decolorized or has only a faint steel-blue color. 



(d) When desired, the specimens may be stained, by way of 

 contrast, with a watery solution of Bismarck brown, dilute fuchsin, 

 saftanin or eosin. 



