STAINING METHODS. 35 
lution will stain sections in four or five minutes. Superfluous color 
is removed by immersing the sections in diluted alcohol or in a one- 
half-per-cent solution of acetic acid for a few seconds. The sections 
are dehydrated in absolute alcohol, cleared up with oil of cedar, and 
mounted in a drop of cedar oil for examination, or in balsam if 
they are to be preserved. 
Gram’s method may be used as directed for cover-glass prepara- 
tions, the sections being first stained in aniline-gentian-violet solu- 
tion (No. 1), then washed in water, or in aniline water as recently 
(1892) recommended by Botkin, then decolorized in the iodine solu- 
tion (see page 29). The sections when decolorized are again washed 
in water, dehydrated in absolute alcohol, cleared in cedar oil, and 
mounted in balsam. 
Weigert’s Method.—This is a modification of Gram’s method in 
which the sections are dehydrated by the use of aniline oil. The 
stained section, after having been washed, is transferred to a clean 
glass slide, the excess of water is removed by the use of filtering 
paper, and the iodine solution is placed upon it in sufficient quantity 
to cover the entire section. When sufficiently decolorized this is re- 
moved in the same way. The section is then dehydrated by placing 
a few drops of aniline oil upon it, removing this with filtering paper, 
and repeating the operation once or twice. The aniline oil must 
then be completely removed by the use of xylol, after which the sec- 
tion is mounted in balsam. 
Kiihne’s Method.—The object of this method is to prevent the removal 
of the color from stained bacteria in sections during the treatment which 
such, sections usually receive before they are ready for mounting—i.e., 
during the washing and dehydrating processes usually employed. For 
staining, Ktihne prefers a methylene-blue solution prepared as follows: 
Methylene blue, 1.5 parts; absolute alcohol, ten parts ; triturate in a watch 
glass and add gradually one hundred parts of a solution of carbolic acid 
containing five parts in one hundred of water. The section is placed in this 
solution for about half an hour, then washed in water and decolorized in a 
weak solution of hydrochloric acid—ten drops to five hundred grammes of 
water. This part of the operation must be conducted very carefully, and 
usually thin sections will only require to be dipped in the acid solution for an 
instant, after which they must be at once immersed in asolution of lithium 
—eight drops of a saturated solution of carbonate of lithium in ten grammes 
of water. They are then allowed to remain in a bath of distilled water for 
a few minutes, after which they are dipped into absolute alcohol, which 
Kiihne colors by the addition of methylene blue. The sections are then 
placed in aniline oil which contains a little methylene blue in solution, 
where they are dehydrated without the color being extracted from thestained 
bacteria present. The aniline-oil blue solution is prepared by adding an ex- 
cess of dry methylene blue to a small quantity of clarified aniline oil. The 
undissolved pigment settles to the bottom, and afew drops of the colored 
solution are added to a little aniline oil in a watch glass to make the colored 
dehydrating bath. The section is next washed out in pure aniline oil—not 
colored—after which every trace of aniline oil is to be removed by the use 
of xylol. The section is cleared up in turpentine and mounted in balsam. 
