STAINING SOLUTIONS 81 
with the stain or immersed in it. For immersion, many different 
types of stainmg jars are available. When the staining procedure 
is carried out by the putting the dye on the bacteria on a slide or cover 
glass, it may be kept in ordinary dropping bottles. 
Stock Solutions of Dyes. These may be made up and kept on 
hand in glass-stoppered bottles. It is best to keep them in the dark 
Such solutions may be made either in alcohol or water. 
Alcoholic Stains. Such stains are made by dissolving a little of 
the dye in alcohol. They are not much used in bacteriology and are 
usually diluted with distilled water. 
Aqueous Alcoholic Stains. These are prepared by adding to dis- 
tilled water a portion of the filtered stock solution of the dye in absolute 
alcohol. These are the stains which have the greatest use in the 
laboratory. From 1 to 5 ¢.c. of the saturated alcoholic solution of the 
dye are diluted with 100 c.c. of the water. 
Aqueous Stains. Pure aqueous solutions are weak stains and have 
lhmited use. They are prepared by adding to the distilled water 
enough of the dye to give a saturated solution. They have the great 
advantage that over staining with them is improbable. 
Mordants. They have the same purpose in staining that they have 
in dyeing. They are substances which unite with the aniline dye and 
with the cell protoplasm. ‘They increase the rapidity of the staining 
procedure and render the color of the stained protoplasm more intense. 
The following substances are used as mordants: 
Iodine in Lugol’s solution. 
Bromine in solution with potassium bromide. 
Tannin. 
Different acids. 
Alkalis. 
Gram Stain. Gram (1884) devised a differential staining procedure 
which has been of much service in bacteriology. The explanation of 
this stain is not definitely settled. Gram in his original work did not 
attempt to explain it. Unna (1887) put the explanation on a chemical 
basis. The Gram stain is an arbitrary procedure and a variation in 
the details of it are very important. It probably depends upon the 
fact that the pararosaniline (methyl violet, gentian violet) compounds 
form substances with iodine which are insoluble in aleohol. The iodine 
salts of rosaniline (fuchsin and methylene blue), however, are decomposed 
by alcohol. The Gram negative bacteria are stained only with the 
gentian violet. The alcohol washes out the iodine. The iodine in 
