STAINING METHODS. 31 
earbol-fuchsin solution (No. 3) the cover glass is placed for one or 
two minutes in a solution containing: 
Sulphurie acid (twenty-five-per-cent solution), : . 100 ce. 
Methylene blue, . Z . P 4 3 2 gms. 
Wash, dry, and mount in cedar oil or balsam. 
METHODS OF STAINING SPORES.— When preparations containing 
the spores of bacilli are stained by any of the methods above given, 
these remain unstained and appear as highly refractive bodies in the 
interior of the rods or filaments in which they have been formed, or 
scattered about in the field if they have been set free. Owing to 
the contrast with the stained protoplasm of the rod or spore-bearing 
filament, they are especially well seen in recent cultures ; while in 
older cultures the bacilli often do not stain well, or are entirely dis- 
integrated and spores only are to be seen. The discovery was made 
at about the same time by Buchner (1884) and by Hueppe that 
spores may be stained if they are first exposed to an elevated tem- 
perature for some time. This may be accomplished by placing the 
slide or cover glass, upon which the spore-containing culture has 
been dried, in a hot-air oven at a temperature of 120° C. for an 
hour; or a higher temperature (180° C.) may be employed for a 
shorter time (fifteen minutes); or the cover glass may be passed 
through the flame of an alcohol lamp or Bunsen burner eight or ten 
times, instead of three times as is customary when the object in 
view is simply to coagulate the albumen and fix the film upon the 
cover glass. After such treatment the spores may be stained with 
an aqueous solution of one of the basic aniline colors—fuchsin, 
methyl violet, etc.—but the bacilli no longer take the stain so well. 
To obtain satisfactory double-stained preparations, showing 
both spores and bacilli, a different method is employed. 
The film upon the cover glass is passed through the flame three 
times, as heretofore directed ; it is then floated upon aniline-fuchsin 
solution in a watch glass, and this is heated to near the boiling point 
for an hour—Neissei’s method. The aniline-fuchsin solution is 
prepared by shaking an excess of aniline oil in a test tube with dis- 
tilled water, filtering the saturated solution into a watch glass, and 
then adding a few drops of a saturated alcoholic solution of fuchsin. 
After this prolonged action of the hot staining fluid the spores of 
some bacilli are deeply stained, while others do not take the stain so 
well. The cover glass is next washed in water and then placed in 
a decolorizing solution containing twenty-five parts of hydrochloric 
acid to seventy-five parts of alcohol. This removes the stain from 
the bacilli, but, if not allowed to act too long, leaves the spores still 
stained. The preparation is next stained in a saturated aqueous 
