1. Xylol. 



2. Canada balsam. 



3. Dammar varnish. 



MICROSCOPIC EXAMINATION. 411 



(D) Clearing and Mounting Agents. 



Preparations of Stained Specimens of Bacteria. 

 (A) Smear Preparations. 



1. Simple Stain with Fuchsin or Methylene-blue. — This may be 

 d for all bacteria except the tubercle bacillus. 

 A. drop of distilled water is placed upon a cover-glass (cover-glass 



preparation) or upon a slide (slide preparation), and in it is mixed a 

 trace of pure culture (preferably from a solid nutrient medium), and 

 the drop is then spread out in a very thin layer. After the fluid has 

 evaporated, the preparation is passed quickly through the flame three 

 times with the film up in order to fix the bacteria to the glass (avoid 

 burning !). The film of bacteria is then covered with the staining solu- 

 tion. After a short time (a few seconds to a minute), perhaps with 

 gentle heat, the preparation is washed with water and allowed to 

 dry (perhaps again with gentle heat). Finally the dry cover-glass is 

 fixed upon a slide by means of a drop of Canada balsam, the bacterial 

 side being down. 



It may here be remarked that slides and cover-glasses upon which 

 the drop of water does not adhere or does not spread out evenly are 

 best cleaned with soap. Alcohol and ether, which are much used for 

 cleaning purposes, do not remove the particles of fat so completely. 



2. Gram's Stain. — 



1. The smear preparation is made as above. 



2. Stain with Ehrlich's solution three minutes. 



3. Wash with water. 



4. Differentiate with Gram's iodin solution one minute. 



5. Decolorize with absolute alcohol until no more color is removed 

 (usually one or two minutes). 



If the alcohol does not decolorize rapidly enough, it can be accele- 

 rated by placing a drop of anilin-xylol upon the preparation and then 

 again washing with alcohol. 



6. Dry and mount. 



According to our experience, the current idea that every variety of 

 bacterium either stains well or not at all when treated by this method 

 is false. Thus, we observed, in the case of the Bact. fluorescens, for ex- 

 ample, which is usually said in the literature not to stain, that three 

 out of twelve different cultures, of twenty-four hours' growth, stained 

 very beautifully. According to Zimmermann, all the varieties of 

 Bact. fluorescens stain in young cultures. 



In like manner we found the bacillus of symptomatic anthrax from 

 a culture to stain, although it has often been said not to stain. The 

 contradictory results may be easily explained in part as due to vary- 

 ing age or youth of materials experimented with, and to variations in 

 the differentiating process with alcohol. The Bacillus tenuis, which 

 has been designated above as failing to stain by Gram's method, upon 



