20() MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
immediately appears. When the precipitate is dry, dissolve it in 
methylic alcohol (Merck's 'reagent') in the proportion of 0.1 gm. to 
60 cc. of the alcohol. In order to facilitate solution the precipitate 
is to be rubbed up with alcohol in a porcelain dish, or mortar with a 
spatula or pestle. 
This alcoholic solution of the precipitate is the staining fluid. It 
should be kept in a well-stoppered bottle because of the volatility of 
the alcohol. If it becomes too concentrated by evaporation and thus 
stains too deeply, or forms a precipitate on the blood smear, the 
addition of a suitable quantity of methylic alcohol will quickly correct 
such faults. It does not undergo any other spontaneous change than 
that of concentration by evaporation. 
"A most important fault met with in the working of some samples 
of this fluid is that it fails to stain the red blood corpuscles a yellow 
or orange color, but stains them a blue color which cannot readily be 
removed by washing with water. This fault is due to a defect in the 
specimen of eosin employed. It can be eliminated by using a proper 
'yellowish, water-soluble' eosin." 
Method of Staini7w. — (a) Unheated air-dried films^ are covered 
with the stain, which is allowed to act for one minute. 
(b) Add an eqiial volume of distilled water to the stain and allow 
to stand for three minutes. 
(c) Wash in water for thirty seconds, or until a pink color develops. 
(d) Dr\' rapidly with filter paper and mount in balsam. ^ 
Giemsa Method.^— Preparation of Stain: 
Azur II (eosin) 3.0 gm. 
Azur II 0.8 gm. 
Glycerin, C. P 250 cc. 
Neutral absolute methyl alcohol 250 cc. 
The dyes are dissolved in the glycerin at 60° C; the alcohol, warmed 
to 40° C, is then added, throughly mixed by shaking, and allowed 
to cool slowly to room temperature, then filtered. Immediately before 
use, 10 cc. of distilled water are slightly alkalinized by the addition 
of 2 drops of a 10 per cent solution of sodium carbonate, and exactly 
10 drops of the stain are then added. 
Staining with (liemsa Solvtions. — {a) Films are fixed by immer- 
sion in neutral absolute methyl alcohol for one minute, air-dried and 
covered with the diluted stain, which is allowed to act for fifteen to 
twenty minutes when ordinary exudates and bacteria are used; for 
one to three hours if Treponemata or Negri bodies are sought for. 
{h) Wash in water, dry and mount. 
The Fontana"^- Trihondeau^ stain for Spirochetes:^ 
' Films more than a few hours old do not stain as well as fresh ones. 
■^ The balsam must be neutral in reaction. 
' Centralbl. f. BakterioL, orig., 1904, 37, .308. 
* Dermatol. Wchnschr., 1913, 56, 301. 
■■' Arch. d. Med. et Pharm., 1913, 90, No. 2. 
« See also Yakimoff: Centralb. f. BakterioL, I Abt., orig., 1927, 102, 89. 
