Staining 153 



cleared in xylol, and mounted in Canada balsam. If it is necessary 

 to study the relation of the bacteria to the tissue elements, a nuclear 

 stain, such as alum-carmin or Bismarck brown, may be previously 

 or subsequently used. Should a nuclear stain requiring acid for 

 its differentiation be desirable, the process of staining must precede 

 the Gram stain, so that the acid shall not act upon the stained 

 bacteria. 



Gram's method rests upon the fact that the combination of bacterial 

 substance, anilin dye, and the iodids forms a compound insoluble in 

 alcohol. 



The process described may be summed up as follows: 



Stain in Ehrlich's anilin-water gentian violet five to thirty minutes; 



Wash in water; 



Immerse two to three minutes in Gram's solution; 



Wash in 95 per cent, alcohol until no more color comes out; 



Dehydrate in absolute alcohol; 



Clear in xylol; 



Mount in Canada balsam. 



No matter how carefully the method is performed, an unsightly 

 precipitate is sometimes deposited upon the tissue, obscuring both 

 its cells and contained bacteria. Muir and Ritchie obviate this 

 (i) by making the staining solution with i 120 aqueous solution of 

 carbolic acid instead of the saturated anilin solution, and (2) by 

 clearing the tissue with oil of cloves after dehydration with alcohol. 

 The oil of cloves, however, is itself a powerful decolorant and must 

 be washed out in xylol before the section is mounted in Canada 

 balsam. 



Gram's method is also employed to aid in differentiating similar 

 species of bacteria in culture. A thin layer of a suspension of the 

 bacteria to be examined is spread upon a slide or cover-glass, dried, 

 and fixed; then flooded with the anilin-oil gentian violet or other 

 staining solution. The solution is kept warm by holding the glass 

 flooded with the stain over a small flame. The process of staining 

 is continued from two to five minutes. If the heating causes the 

 'stain to evaporate, more of it must be added so that it does not 

 dry and incrust the glass. 



The stain is poured off, and replaced by Gram's solution, which 

 is allowed to remain from one-half to two minutes, and gently 

 agitated. 



The smear is next washed in 95 per cent, alcohol until the blue 

 color is wholly or almost lost, after which it can be counterstained 

 with pyronin, eosin, Bismarck brown, vesuvin, etc., washed, dried, 

 and mounted in Canada balsam. Given briefly, the method is: 



Stain with Ehrlich's solution two to five minutes; 



Gram's solution for one-half to two minutes; 



Wash in 95 per cent, alcohol until decolorized; 



Counterstain if desired; wash off the counterstain with water; 



Dry; 



Mount in Canada balsam. 



