1887. | MICROSCOPICAL JOURNAL. 227 
MICROSCOPICAL TECHNIQUE. 
Staining of schizomycetes in sections and dry preparations.* 
C. Gram proposes the following method of producing an isolated stain- 
ing of pneumonia-cocci, leaving the nuclei and other elements of the tissue 
uncolored, the deep staining of the cocci usually found in the several shells 
causing them to be more readily found than in ordinary preparations. The 
method he considers applicable also to almost all examinations of schizomy- 
cetes, in sections and dry preparations. ; 
He takes the ordinary Erlich’s anilin-gentian-violet solution. The sections 
to be examined for schizomycetes must be preserved in absolute alochol, and 
brought direct from it to the staining fluid; here they remain from I-3 min- 
utes (in the case of preparations of tubercular bacilli from 12-24 hours) ; 
they are then placed in an aqueous solution of potassium biniodide (1 part I, 
2 parts KI, 300 parts water), without or after a slight washing with alcohol, 
where they remain again from 1-3 minutes. A precipitate takes place in the 
iodine solution, and the sections, previously a dark blue-violet, become a 
blackish purple-red. They are now laid again in absolute alcohol until the 
color is again entirely removed, the alcohol being renewed once or twice. They 
are then clarified in the ordinary way by clove oil, the remainder of the pig- 
ment being given off to the oil. The nuclei and the fundamental tissue are 
now colored light yellow by iodine, while the schizomycetes, if present in a 
section, are of a conspicuous intense blue color, often nearly black, the color 
being much deeper than in any other mode of staining. After the application 
of alcohol the sections may be placed for a moment in a weak solution of Bis- 
marck brown or vesuvin, in order to produce a double-stain. 
Permanent preparations have been kept for four months without change in 
Canada balsam, hylol, or gelatin glycerin. The whole of the process takes 
a quarter of an hour, and the preparations may remain for some days in clove 
oil without losing their color. The method can also be applied to dry prep- 
arations, the cover-glass being treated as a section. The following tissues 
were tested for schizomycetes by this method :—Pneumonia cruposa, pyemia, 
nephritis suppurativa, arthritis suppurativa after scarlatina, multiple brain 
diseases, osteo-myelitis, typhus, liver abscesses, erysipelas, tuberculosis, cat- 
tle distemper, as well as the bacteria of putrefaction. After treatment with 
iodine the following schizomycetes remained colored in alcohol :—The cocci 
_ of crupose pneumonia, the schizomycetes of pneumonia, the cocci of the liver 
abscesses after perityphlitis, the cocci and small bacilli in circumscribed infil- 
tration of the lungs, the cocci of osteo-myelitis, of arthritis suppurativa after 
scarlatina, of nephritis suppurativa after cystis, those of multiple brain ab- 
scesses, of erysipelas, the bacilli of tubercular cattle distemper, and the schizo- 
mycetes of putrefaction. On the other hand no staining was exhibited of the 
capsular cocci in a case of crupose pneumonia, or of the capsules without 
cocci in another case, or of the bacilli of typhus. 
Staining bacillus tuberculosis. 
The following modification of Ehrlich’s method of staining Baczllus tuber- 
culosis was devised by Dr. Heneage Gibbes :— 
‘(1.)¢ Take 2 grms. of magenta crystals, 3 grms. of pure anilin, 20 cc. of 
alcohol (specific gravity .830), 20 cc. of distilled water. Dissolve the anilin 
* Fournal Royal Microscopical Society for 1884, p. 817-8. 
+‘A Synopsis of the Bacteria and Yeast Fungi, and allied species,’ by W. B. Grove, p. 103-4. 
