574 
Journal of Agricultural Research 
Vol. XIV, No. 13 
fore, with a view to overcoming the several obstacles attending the 
preparation of the trypanosomal antigen, the following technic has 
been devised and has given good results. 
TECHNIC OF NEW METHOD 
Blood of infected rats is collected in a 1 per cent sodium-citrate solution 
in physiological salt solution in order to prevent coagulation. When 
all the blood has been collected, the solution is filtered through cheese¬ 
cloth to remove clots, fibrin, etc., poured into tubes, and centrifugalized 
for about 20 minutes at 2,100 revolutions per minute. This precipitates 
all the corpuscles and most of the trypanosomes, leaving an upper 
stratum of blood serum and citrate solution containing some of the organ¬ 
isms. This fluid is drawn off and again centrifugalized in order to recover 
any of the protozoa which may be present. To the other tubes containing 
the mass of corpuscles intermixed with and superimposed by trypano¬ 
somes is added sufficient distilled water to produce complete hemolysis 
of the rat erythrocytes, a matter of about 20 minutes, which procedure 
is facilitated by agitation of the mixture in a flask. This also is cen¬ 
trifugalized but, in this instance for about half an hour, upon the com¬ 
pletion of which there is found at the bottom of the tubes a mass of 
trypanosomes with an admixture of stroma of the hemolyzed red cells, 
which latter, in quantity, has been found to be negligible. After dis¬ 
carding the supernatant fluid (hemoglobin-stained water) physiological 
salt solution is added and the material vigorously shaken until the mass 
of trypanosomes is disintegrated and evenly distributed throughout the 
solution. Centrifuging is again resorted to with similar results, the 
washed mass of trypanosomes being packed at the bottom of the tubes. 
The salt solution is poured off and an amount of preserving fluid 
(physiological salt solution and glycerin aa) equal to about twice the 
amount of trypanosomes added; the mixture is then agitated until a 
uniform suspension is acquired, when it is stored at a low temperature 
until used. 
In order to determine whether the use of distilled water in laking the 
corpuscles would have any detrimental effect on the trypanosomes as 
regards their antigenic value, immediately following their preparation, 
and also after an interval of two weeks, the following procedure was 
employed: 
Twenty white rats were inoculated subcutaneously with a suspension 
of Trypanosoma equiperdum in salt solution. At the end of the third 
day the blood of all the animals showed a heavy infestation with the 
protozoa. 
The animals were then bled to death into 300 cc. of a 1 per cent sodium- 
citrate solution in physiological salt solution, Vhich was then divided 
equally and placed into two flasks. 
