28 LABORATORY BACTERIOLOGY 
39. Making aniline water. Aniline water is a Saturated 
aqueous solution of aniline oil. It is prepared by adding 1 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 1 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 1 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) 
Jodineé 2. 2 on ee we He Ke HH a a Se E gram 
Potassiumiodide . . . ... . . . + «. 2grams 
Distilled water . 2. 2... 1 ew ee es 300 CE, 
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. 
