28 LABORATORY BACTERIOLOGY 



39. Making aniline water. Aniline water is a saturated 

 aqueous solution of aniline oil. It is prepared by adding i cc. 

 of aniline oil to 20 cc. of distilled water and shaking frequently 

 for from 15 to 30 minutes. It is convenient to use a stoppered 

 vial or large test tube for mixing it. Filter through a mois- 

 tened filter paper. The filtrate should be perfectly clear. If 

 it is cloudy, it should be refiltered before using. This is used 

 in preparing the aniline water dyes, such as methyl violet, 

 gentian violet, etc. 



40. Gram's method of staining bacteria. Prepare the cover- 

 glass preparations as already described. Stain them in gentian- 

 violet aniline water, or in a saturated alcoholic solution of 

 gentian violet in 5 % carbolic acid in the proportion of i to 20 

 for from 5 to 7 minutes. Rinse in water and transfer them to 

 a watch glass containing Gram's solution until the color becomes 

 quite black. This requires from i to 2 minutes ; then place 

 the preparations in a watch glass containing alcohol and allow 

 them to remain there until the color has almost entirely dis- 

 appeared, or has become a pale gray. Rinse in water and 

 examine at once, or allow them to dry and mount in balsam. 

 (Sections of tissues must be dehydrated and cleared before 

 mounting.) 



GRAM'S SOLUTION (LUGOL'S) 



Iodine i gram 



Potassium iodide 2 grams 



Distilled water . . 300 cc. 



Certain bacteria stain deeply and retain the coloring matter 

 when treated by this method, while others are decolorized by 

 the alcohol. On this account some investigators consider it an 

 important aid in the differentiation of certain bacteria. 



