METHODS OF OBSERVING BACTERIA. 103

will appear. Ix>ffler has fortunately worked out the
amounts required for some of the species, and of the
more important ones the following amounts of Solutions
B and C must be added to 16 c.cm. of Solution A to
attain the desired effect:

Cholera spirillum, j£-i drop of Solution C ;

Typhoid fever, I c.cm. of Solution B ;

Bacillus subtilis, 28-30 drops of Solution B ;

Bacillus of malignant edema, 36-37 drops of Solution B.

Part of the success of the staining depends upon
having the bacteria thinly spread upon the glass, and as
free from albuminous and gelatinous materials as possi-
ble. The cover-glass must be cleaned most painstakingly :
too much heating in fixing must be avoided. After using
and washing off the mordant, the preparation should be
dried before the application of the anilin-water-fuchsiu
solution.

Pitfield1 has devised a simple and good method of
staining flagella. A single solution at once mordant and
stain is employed. It is made in two parts, which are
filtered and mixed.

A. Saturated aqueous solution of alum, 10 c.cm.;
Saturated alcoholic solution of gentian-violet, i c.cm,

B. Tannic acid, i gr.;
Distilled water, 10 c.cm.

The solutions should be made with cold water, and
immediately after mixing the stain is ready for use. The
cover-slip is carefully cleaned, the grease being burned
off in a flame. After it has cooled the bacteria are
spread upon it, well diluted with water. After drying
thoroughly in the air, the stain is gradually poured on
and by gentle heating brought almost to a boil; the slip

1 Med. News, Sept. 7, 1895.