32 STAINING METHODS. 
solution of methylene blue; and if the operation has been successfully 
carried out the spores will be stained red and the protoplasm of the 
bacilli in which they are present will be blue. : 
Moller has (1891) published the following method of staining 
spores : 
The cover-glass preparation, dried in the air, is passed three times 
through a flame or placed for two minutes in absolute alcohol; it is 
then placed in chloroform for two minutes and washed in water; it 
is now immersed in a five-per-cent solution of chromic acid for from 
half a minute to two minutes and again thoroughly washed in 
water; next a solution of carbol-fuchsin is poured upon it and it 
is heated over a flame until it commences to boil, for sixty seconds; 
the carbol-fuchsin solution is then poured off and the cover glass is 
immersed in a five-per-cent solution of sulphuric acid until the 
film is decolorized, after which it is again thoroughly washed in 
water. It is then placed for thirty seconds in an aqueous solution of 
methylene blue or of malachite green, and again washed in water, 
after which the preparation should be dried and mounted in balsam. 
As a result of this procedure the spores are stained dark red and the 
protoplasm of the bacilli blue or green. 
Fioccu (1893) claims that better results are obtained by the follow- 
ing method: 
About twenty cc. of a ten-per-cent ammonia solution is placed in a 
watch glass, and from ten to twenty drops of an alkaline solution of 
an aniline color is added; heat is applied until steam commences to be 
given off, when the cover glass is placed in the hot solution for from 
three to fifteen minutes. The cover glass is then quickly washed in 
a twenty-per-cent solution of nitric or sulphuric acid to decolorize; 
then it should be thoroughly washed in water, after which it may 
be stained with a contrast color by the use of an aqueous solution of 
one of the aniline dyes—preferably vesuvin, malachite green, or 
safranin. 
METHODS oF STAINING FLAGELLA.—Koch first succeeded in de- 
monstrating the flagella of certain bacilli and spirilla by staining 
them with an aqueous solution of hematoxylon, and dilute chromic 
acid asa mordant. Léffler (1889) has succeeded in demonstrating, 
by an improved staining method, the presence of flagellain a consider- 
able number of species in which they had not previously been seen, 
although generally suspected to be present. His method is as follows: 
Léffler’s Method.—The following solution is used as a mordant: 
No. 1. 
Solution of tannin of twenty per cent, : A 10 ce. 
Saturated (cold) solution of ferrous sulphate, . ‘ . Bee 
Aqueous or alcoholic solution of fuchsin, 1ce. 
(Or one cubic centimetre alcoholic solution of methyl violet.) 
