MICHIGAN ACADEMY OF SCIENCE. 
145 
THE FLAGELLUM STAINING OF SPIROCHAETA OBERMEIRL 
CLOUGH TURRILL BURNETT. 
To demonstrate the flagellum of Spirochaeta Obermeiri it is neces¬ 
sary to separate the spirochaetes from the blood containing them. The 
parasites are injected into the peritoneum of a white rat and appear in 
the blood two days later. This blood is drawn and allowed to stand, 
from eighteen to twenty-four hours. At tire end of that time the blood 
cells will have settled to the bottom of the tube and above these there 
is a thin whitish layer of spirochaetes. Attempts were made to stain 
spreads made from this material, but this proved unsuccessful, as the 
blood plasma present masked the whips. To prevent this the plasma 
is drawn off with a sterile pipette, care being taken not to disturb the 
layer of spirochaetes, and its volume replaced by a normal salt solution. 
This tube is now sealed and centrifuged for twenty to thirty minutes and 
then is opened, the supernatant fluid removed and more salt solution 
put in, and after sealing again centrifuged. It may be necessary in 
some cases to repeat this operation three or four times, examinations 
being made each time the tube is opened. When in the hanging drop a 
clear field is obtained with only a few blood cells and many motile 
spirochaetes, spreads are made, dried in air and fixed five minutes in 
methyl alcohol. If agglutination is taking place there may appear on 
the surface of the layer of blood cells small whitish “islands” of 
spirochaetes. One of these islands can be picked up with a fine pipette 
and placed in suspension in a few drops of normal salt solution. If 
examination shows that there are still many motile spirochaetes present 
spreads may be made directly from this without centrifuging, for by 
this method there will not be enough blood plasma present to mask the 
whips. 
The fixed cover-glasses are now treated with a hot mordant solution 
and then with a stain. 
The mordant solution is that used by Duckwall slightly modified. Its 
composition is as follows: 
Dry tannic acid—2 grams. 
Distilled water—15 c. c. 
Ferrous sulphate (cold water sol.)—4 c. c. 
Fuchsin (saturated alcohol sol.)—1 c. c. 
The tannin is added to the water and heated; then add the ferrous 
sulphate and last the fuchsin. Filter through a fine filter. There should 
be a thick heavy precipitate on the filter. The filtrate, which is the 
mordant solution, is a cloudy violet. This is good for not more than 
five hours and is best when fresh. 
The stain is an anilin water fuchsin. To 100 c. c of a hot anilin 
water is added four to five grams of fuchsin. Stir well until dissolved, 
and filter. It has been found that the best results in flagella staining 
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