‘ 
1887.] ‘MICROSCOPICAL JOURNAL. 193 
ular cases, and the investigation staining used especially to prove the pres- 
ence of bacteria. . 
Since in cover-glass preparations almostall bacteria can be stained by watery 
solutions of the basic anilin dyes, saturated watery solutions, or the equally valu- 
able alcoholic solutions, are first employed. The saturated watery solutions 
have for this testing an advantage, becaus all basic anilin dyes are known 
to be applicable, so, with a few preparations, the different colors can be tried. 
If no bacteria come to view in this way, notwithstanding their supposed 
presence, then anilin water, with methyl violet or fuchsin, is used, or the 
stronger alkaline solution of methyl blue.* The trial examination as to the 
presence of bacteria resolves itself, in the larger number of cases, into the 
following procedure :— 
1. Drying in a thin layer. 
2. Fixation by passing the cover-glass three times through the flame. 
3. Staining by placing a few drops of a watery or dilute alcoholic solution 
of a basic anilin dye upon the preparation. 
4. Removal of the excess of the coloring-matter by washing or soaking up 
with filter-paper. 
5. Examination in a drop of distilled water. 
For the isolated staining of bacteria in cover-glass preparations they can be 
laid for about one minute in a half-saturated solution of potassium carbonate, 
or, if they are stained in anilin-water gentian-violet, the remaining elements 
can be decolorized according to the method of Gram. The stained cover-glass 
preparations are, for this purpose, laid for about one minute in a solution of 
potassium iodine (iodine 1 part + potass. iodid. 2 parts + water roo parts) 
and then placed in absolute alcohol until they appear decolorized. The alco- 
hol is soaked out and the preparations examined in water. 
For double-staining the cover-glass preparations, after decolorizing accord- 
ing to the Gram method, they can be taken from the alcohol and placed in a 
weak watery solution of yesuvin. Then the bacteria remain blue, often almost 
blue-black, while the nuclei are stained brown. The preparations stained red 
or blue can also afterward be stained with carmine or hematoxylin, yet this 
double-staining has much less value in the cover-glass preparations than in 
sections. 
EXAMINATION FOR TUBERCLE BACILLI IN SPUTUM. 
These preparations can be stained according to the Gram method ; but by 
this both the tubercle bacilli and other bacteria are stained blue in contrast 
with the brown nuclei. For the differential diagnosis this is not sufficient, 
and for.this purpose the principle established by Koch must be exclusively ob- 
served, z. e., that the tubercle bacilli should be stained in a different color 
from other bacteria and the nuclei. Koch succeeded in doing this in prep- 
arations stained for twenty-four hours in a weak solution of methyl-blue, and 
then placed for a short time in a watery solution of vesuvine. In this way 
the tubercle bacilli (and the bacilli of leprosy) are stained blue; all other 
bacteria and nuclei brown. After this important principle was discovered, 
Ehrlich showed that anilin-water+ was a still better method for increasing the 
intensity of the color, and that in the preparations stained with analin water the 
tubercle bacilli withstood decolorization by nitric acid, while all other bacteria 
were decolorized by this minéral acid. But the preparations cannot be left so 
long in the acid that complete decolorization occurs, because then also many 
* Concentrated alcoholic solution methyl blue . . . . . 30C.c. 
Solution caustic potash, 1to1,o0o0, . .. . =. . =. . TOOC.C. 
+ Pure anilin oil in excess (about 5 c.c. of oil and 100 c.c. water) is shaken with distilled water for one-half 
to one minute. Then, after allowing it to stand five minutes, the mixture is filtered. The filtrate must be per- 
Acctly clear, and serves in place of water asa menstruum. It is very unstable, and should be prepared fresh 
each time. 
