METHODS OF OBSERVING BACTERIA. 83 



Friedlander recommends that the section remain from 

 fifteen to thirty minutes in warm stain, and in many cases 

 the prolonged process gives better results. 



From the stain the section is given a rather hasty wash- 

 ing in water, and then immersed from two to three min- 

 utes in Gram's solution (a dilute Lugol's solution) : 



lodin crystals, i ; 



Potassium iodid, 2 ; 



Water, 300. 



While the specimen is in the Gram's solution it 

 appears to turn a dark blackish-brown color. When 

 removed from the solution it is carefully washed in 95 

 per cent, alcohol until no more color is given off and 

 the tissue assumes a grayish color. If it is simply 

 desired to find the bacteria, the section is dehydrated 

 in absolute alcohol for a moment, cleared up in xylol, 

 and mounted in Canada balsam. If it is necessary to 

 study the relation between the bacteria and the tissue- 

 elements, a nuclear stain, such as alum carmin or Bis- 

 marck brown, may be subsequently used. Should a 

 nuclear stain requiring acid for its differentiation be 

 desirable, the process of staining must precede the Gram 

 method altogether, so that the acid shall not act upon 

 the stained bacteria. 



The success of Gram's method rests upon the fact that 

 the combination of mycoprotein, basic anilin, 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 momentarily 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 up in xylol ; 



Mount in Canada balsam. 



