METHODS FOR MICROSCOPIC STUDY OF BACTERIA 199 
2. The film of bacteria is allowed to dry in the air; evaporation 
may be hastened in the incubator at 37° C. 
3. The air-dried film is next fixed by passing it once slowly through 
the flame of a Bunsen burner, film side upward; above one-half second's 
exposure to the flame suffices; a longer exposure destroys or changes 
the staining properties of the organisms. 
4. Staining: A 5 per cent aqueous solution of methylene blue, 
fuchsin, or gentian violet, prepared by adding 5 cc. of filterefl satu- 
rated stock solution to 95 cc. of distilled water, is used. The slide 
or cover-glass is flooded with the desired stain, and after one to five 
minutes, depending upon the intensity of the stain employed, the excess 
is poured off and the preparation is washed thoroughly wdth water. 
The residual moisture is removed with, filter paper or by air-drying. 
A small drop of Canada balsam (dissolved in xylol) is placed in the 
center of the stained area, and covered with a cover-glass if a permanent 
preparation is desired. Neutral Canada balsam should be used. 
D. Intensive Stains for Bacteria.— Simple aqueous or alcoholic solu- 
tions of anilin dyes are frequently inefficient for staining bacteria and 
resort is made to intensified stains. One of the most useful of the 
intensified stains is Loffler's alkaline methylene blue, prepared in the 
following manner. 
1 to 10,000 aqueous solution of potassium hydroxide' 70 cc. 
Saturated alcoholic solution methylene blue 30 cc. 
Fixed films of bacteria are stained from one to five minutes with 
this stain, or the films are flooded with the stain and heated until 
steam rises (not boiled) for one to three minutes. It is difficult to 
overstain with Loffler's methylene blue unless evaporation takes 
place to such a degree that the stain dries on the slide. The stain is 
washed off with water, and the preparation is dried and mounted. 
E. Stains for Special Structures of the Bacterial Cell. — 1. Spores.— 
(a) Flood fixed film of bacteria with carbol-fuchsin^ and steam (not boil) 
for five minutes. 
(b) Wash thoroughly in running water. 
(c) Decolorize with 1 per cent sulphuric acid until the excess of stain 
is removed. 
{(}) Wash thoroughly in running water. 
(e) Flood with saturated aqueous solution methylene blue (or 
Loffler's alkaline methylene blue) and allow to stain one minute. 
(/) Wash in water, dry and mount. 
Spores stain red, vegetative cells blue. 
Mdller's Spore Stain. ^—Suspend the fixed film of bacteria in chloro- 
form for two minutes. 
1 Conveniently prepared by dissolving 1 gram of KOH in 100 cc. of distilled water 
and adding 1 cc. of this solution to 99 cc. of distilled water. 
'^ Saturated alcoholic solution of basic fuchsin, 10 cc; 5 per cent aqueous phenol 
solution, 90 cc. 
3 Centralbl. f. Bakteriol., 1891, 10, 273. 
