146 Methods of Observing Bacteria 



The specimen while in the Gram solution turns a dark 

 blackish-brown color, but when removed and carefully 

 washed in 95 per cent, alcohol again becomes blue. The 

 washing in 95 per cent, alcohol is continued until no more 

 color is given off and the tissue assumes its original color. 

 If it is simply desired to find the bacteria, the section can 

 be dehydrated in absolute alcohol for a moment, 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 mycoprotein, anilin dye, and the iodids forms a compound 

 insoluble in alcohol. 



The process described may be summed up as follows: 



Stain in Khrlich'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. 



This method stains the majority of bacteria, but not all, 

 hence can be used to aid in the differentiation of similar 

 species. Some bacteria that do not stain are important : 



Spirillum cholerae asiaticae ; 



Spirillum cholerae gallinarum ; 



Bacillus mallei (of glanders) ; 



Bacillus oedematis maligni ; 



Bacillus pneumoniae (Friedlander) ; 



Micrococcus gonorrhoeas (Neisser) ; 



Spirochaete obermeieri (of relapsing fever) ; 



Bacillus typhosus; 



Bacillus dysenteriae; 



Bacillus suipestifer ; 



Bacillus coli communis; 



Bacillus icteroides; 



Bacillus of rabbit septicemia ; 



