8 
Journal of Agricultural Research 
Vol. VIII, No. i 
finally kept in a well-stoppered vial in a cool place protected from the 
light. 
Standardization or Antigen for Final Test 
Complement. —In the early work of the writers on the complement fixa¬ 
tion in tuberculosis the intention was to use one unit of the accurately 
standardized complement. The titer was determined by establishing the 
smallest amount of complement which caused complete hemolysis 
within one-half hour of incubation. It was soon observed, however, 
that this amount was insufficient to establish the proper amount of 
complement necessary to obtain the best results. It was found that a 
5 per cent dilution proved most suitable, and accordingly that propor¬ 
tion was employed throughout this work. 
Two units of anti-sheep-blood hemolysin were used throughout. 
Red sheep-blood corpuscles. —Various dilutions of the suspension 
were tested, and the most satisfactory results were obtained with a 
3.5 per cent sheep-blood corpuscle. 
Salt solution. —While the normal physiologic salt solution was used 
in most cases, tests were also made with 0.75 per cent. It was noted 
that by employing the hypotonic solution the reactions developed more 
rapidly. 
Serums used for standardization of antigens. —The serums were 
obtained from tuberculin-tested animals. At least 12 months elapsed 
from the time of the last negative test. The inactivation was carried 
on at 58° C. for one-half hour and 0.2 c. c. was the amount used in the 
test in every tube. 
Two methods were used in the standardization of the antigen. 
method i: standardization of antigen 
Thirty mgm. of defatted and dried tubercle bacilli were pulverized 
in a 12-cm. mortar. The powder was then emulsified in a solution 
containing 50 mgm. of dried tuberculin in 10 c. c. of 0.6 per cent salt 
solution. This emulsion served as the antigen and was titered for its 
antigenic, anticomplementary, and hemolytic properties. The titration 
was conducted according to Tables I to III. 
Table I .—Titration for antigenic dose 
Tube 
No. 
Antigen. 
Inacti¬ 
vated 
positive 
serum. 
Comple¬ 
ment, 5 
per cent. 
Physio¬ 
logical 
salt so¬ 
lution. 
Incu¬ 
bation 
period. 
Hemolysin. 
Erythro¬ 
cytes, 
3 5 Per 
cent. 
Incu¬ 
bation 
period. 
Degree of 
hemolysis. 
C. c. 
C. c. 
C. c. 
C. c. 
1 
0.5 
0.2 
1 
'll O 
2 units con¬ 
1 
None. 
S A 
tained. 
2 
*4 
. 2 
1 
2 w 
.do. 
1 
Do. 
3 
• 3 
. 2 
1 
.do. 
1 
Do. 
4 
■ 2 
. 2 
1 
-a *8 
§ 
.do. 
1 
g 
Do. 
5 
. 1 
. 2 
1 
la 
’ JS 
.do. 
1 
. A 
Incomplete. 
6 
• 05 
. 2 
r 
S 0 
HI 
.do. 
1 
H 
Complete. 
7 
None. 
. 2 
1 
.do. 
1 
Do. 
8 
. 1 
None. 
1 
.do. 
z 
Do. 
