Staining 163 



Micrococcus melitensis; 

 Spirillum cholerse asiaticae; 

 Spirillum cholerae nostras; 

 Spirillum cholerae gallinarum; 

 Spirillum metschnikovi; 

 Spirillum tyrogenum; 

 Spirochaete duttoni; 

 Spirochaete obermeieri; 

 Spirochaete pertenuis; 

 Spirochaete refringeus; 

 Treponema pallidum. 



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 : 20 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 differentiat- 

 ing 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 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. 



