MICROSCOPIC METHODS 69 



smear will he left on them. If it is desired to preserve the blood cells 

 intact the films are placed in a saturated solution of corrosive sublimate 

 for two to three minutes, and then washed in running water, or instead 

 of sublimate exposed to the vapor of formalin. 



When milk films are made they are, after fixation, cleared of fat by 

 means of ether. From whatever source derived the film is allowed to 

 dry thoroughly at the usual air temperature, and then, in order to fix 

 the film with its contained bacteria to the glass, the latter is grasped in 

 any one of the several kinds of forceps commonly used, and is passed 

 three times by a rather slow movement through the Bunsen or alcohol 

 flame. Instead of this method the film may be fixed to the glass by 

 placing it in absolute alcohol for a few minutes. The smear thus pre- 

 pared is usually stained either by the simple addition of a solution of 

 an aniline dye, for from one to five minutes, or by one of the more 

 complicated special stains described later. When the stain is to be 

 hastened or made more intense the dye is used warm. For ordinary 

 staining the bacteria are simply covered completely by the cold staining 

 fluid. 



The cover-glass or slide, with the charged side uppermost, may 

 either rest on the table or be held by some modification of Cornet's 

 forceps. When the solution is to be wanned the cover-glass may be 

 floated, smeared side down, upon the fluid contained in a porcelain 

 dish resting on a wire mat, supported on a stand, or it may be held in 

 the Cornet forceps. If a slide is used it is simply inserted in the fluid 

 or covered by it. The fluid in both the dish and on the glass should 

 be carefully warmed, so as to steam without actually boiling. The 

 glass should be kept completely covered with fluid. The bacteria 

 having now been stained, the cover-glass or slide is grasped in the 

 forceps and thoroughly but gently washed in clean water and then 

 dried, first between layers of filter-paper and then in the air or high 

 over a flame. A drop of balsam or water is now placed on a glass slide 

 and the cover-glass placed upon it with the bacterial side down. The 

 cover-glass or slide preparation is now ready for microscopic exami- 

 nation. 



Staining of Bacteria. The protoplasm of bacteria reacts to stains 

 much as nuclear chromatin, though sometimes more and sometimes 

 less actively. 



The best stains are the basic aniline dyes, which are compounds 

 derived from the coal-tar product aniline (C 6 H 5 XH 2 ). 



AXILIXE DYES. The aniline dyes which are employed for staining 

 purposes are divided into two groups according as the staining action 

 depends on the basic or the acid portion of the molecule. The former 

 contains amido groups and are spoken of as nuclear stains, since they 

 color the nuclear chromatin of both cells and bacteria. The latter 

 contain hydroxyl groups and do not stain bacteria, but are used chiefly 

 for contrast coloring. The basic dyes are usually employed as salts 

 of hydrochloric acid, while the acid dyes occur as sodium or potassium 

 salts. 



