146 
EIGHTH REPORT. 
are obtained when a weak alkali is added, so to this dye should be added 
1 c. c. of a 1% NaOH solution. As both the mordant and the stain are 
used hot, these may be placed on a metal plate, one end of which is 
heated by a Bunsen burner, or they may be heated over a water bath. 
The fixed cover-glass is now treated with the hot mordant. Before 
applying this it is best to cover the cover-glass with distilled water 
and heat to steaming, then put on the hot mordant. This prevents that 
precipitation which occurs when the hot mordant is placed on a cold 
cover-glass. Since in heating this mordant there is some precipitation, 
care should be taken not to boil it on the cover-glass. After treating 
with the steaming mordant solution for two minutes, wash under the 
tap until all deposit is removed. Again cover with distilled water and 
heat to steaming; then apply hot anilin water fuchsin for two and a 
half minutes, boiling gently at the end. Wash) under tap, then with 
dil. alcohol and absolute alcohol until no more dye can be removed. 
Finally dip for one to two seconds in dilute HNO s (5 drops of C. P. acid 
to 10 c. c. water). If the cover-glass is transferred directly from alcohol 
to acid it should be removed as soon as possible and washed with water, 
as in this case we get the action of an acid alcohol which is a much 
stronger decolorizing agent. If the specimen is washed in water after 
being taken from alcohol, a longer time will be required in the acid. 
The specimen should be examined from time to time to make sure that 
decoloration is not carried too far. This procedure will show deeply- 
stained spirochates on an almost colorless background and may show 
some very faint terminal whips. If these are present they will prob¬ 
ably be very hard to see, but by repeating the above procedure in its 
entirety, that is, by again treating with mordant and stain and decolor¬ 
ing carefully, a very fine terminal whip will be seen to stand out plainly. 
This whip is spiral in shape like the body of the cell, and by this- 
method of staining appears to be about one-third as thick as the body 
of the spirochaete. It may be composed of from one to six turns; in 
one case a whip was found which was longer than the body of the cell. 
At the other end is a short stub which may mark the beginning of the 
formation of a flagellum. This blunt process does not appear in all 
forms which show a flagellum. Many organisms are found to be at¬ 
tached by their whips. 
In my work thus far I have been unable to stain these whips without 
considerable precipitation, so that probably in many cases the whips 
have been masked by a heavy precipitate. This precipitate could be 
washed out with alcohol and acid, but in so doing the few whips present 
in these cases were decolorized. Also much care must be exercised in 
the complete removal of the blood plasma. 
At present I am attempting to stain these whips by using this same 
mordant and dye, but at a constant temperature of 70°—77° C. The 
mordant and dye are made up at a temperature of 55°—60° 0., so 
by this rise in temperature I am assured of complete solution of my 
dye and thus, I believe, reduce materially the precipitation. 
This latter work has not progressed sufficiently far to warrant any 
report. 
University of Michigan. 
