E. A. Watson 
163 
clots and fibrin into narrow centrifuge tubes, 10 mm. diameter and 
10 c.c. capacity (when wider tubes are used it is more difficult to separate 
the trypanosomes and the wastage is greater). Centrifuge not longer 
than four to five minutes at 1500 revolutions per minute so that the 
bulk of the corpuscles are thrown down while the trypanosomes remain 
in suspension. Draw off the cloudy suspension fluid into fresh tubes, 
then the upper layer of corpuscles—more or less mixed with trypano¬ 
somes—into another tube, and the next layer into a second tube, 
adding citrated salt solution and again centrifuging for 8-10 minutes. 
Draw off and discard as much of the upper fluid as appears clear and 
free from trypanosomes. Then collect from each tube into a single 
tube the upper pure white layer of trypanosomes, in another tube the 
middle layers slightly soiled with blood, and in a third and fourth tube 
the lower layers in contact with the blood cells. Add normal salt 
solution now, not citrate, shake up well and centrifuge again, repeating 
the washings until all the trypanosomes are obtained in a pure white 
mass. 
Ten rats bled at the right time will furnish 5-0 c.c. of trypanosomes. 
Twice the volume of the glycerine-formalin preservative is added and 
the mixture stored in sealed amber ampoules, 1-0 c.c. in each, in a block 
of ice. 
5-0 c.c. of trypanosomes will make 100 c.c. of antigen, sufficient for 
more than 500 diagnostic tests. The antigen will keep indefinitely if 
solidified by freezing, and for 6-8 weeks or longer when stored in liquid 
form, in sealed ampoules, on ice. 
(y) Antibody. In the diagnostic tests the antibody, of course, is or 
is not present in the suspected test serum. But for purposes of control 
and titration and to thoroughly understand the combining action of 
dourine antigen and antibody it is absolutely necessary to have one or 
more series of known positive or specific dourine horse sera, of which 
the antibody content can be determined. To obtain this a horse is 
inoculated with Trypanosoma equiperdum. Ten days later and at weekly 
intervals thereafter, blood is drawn aseptically from the jugular vein, 
the serum collected and tested for antibody content (vide p. 173). 
A series of specific positive sera are thus obtained, representative of 
different periods and stages of the disease. Stored in the ice chamber 
the sera will retain their specific properties for many months, even years, 
if collected sterile. If not absolutely sterile the serum may be preserved 
by adding 1-0 c.c. of 5 per cent, carbolized glycerine, or the same amount 
