ANTHRAX 105 
The quality of the immune serum is roughly estimated as follows: The serum 
that will produce a reaction immediately when brought into contact with an anthrax 
antigen is considered univalent. It has been learned that good sera will show the 
presence of an anthrax antigen in a normal unit and, also, in fractions of the same. A 
serum that reacts to an antigen diluted 100 times is a hundred fold serum. This is 
called a standard serum. 
In testing the material for the presence of anthrax by the Ascoli reaction, it is not 
known whether the precipitinogen is present or not, hence its titration is impossible. 
But in case of the titration of the immune serum where the antigen is prepared from 
known anthrax material, some kind of a standard is necessary. For this standard, the 
culture extract is used. 
TECHNIC OF THE TEST 
According to Ascoli, the serum should first be placed in a test tube after which the 
extract, the lighter of the two fluids, should be placed beneath it by means of a capillary 
pipette. According to Schutz and Pfeiler, just as good results are obtained by placing 
the heavier fluid, the immune serum, in the tube first and then placing the antigen on 
top of it. They use thesame technic used by Pfeiler in his diagnosis of glanders by the 
precipitation reaction. With an ordinary pipette, they place the serum drop by drop 
on the edge of a small test tube, about 6 millimeters in diameter and 12 centimeters 
long. The serum is allowed to run slowly down the inside of the tube to the bottom. 
Enough serum should be added to bring the top of the fluid up about one-half a centi- 
meter in height. After the serum has reached the bottom and remains stationary, the 
antigen should be placed on the edge of the tube in the same manner as the serum, and 
allowed to run slowly down the tube until it reaches the serum. It is advantageous to 
have the antigen take the same course down the tube as the serum. When the extract 
reaches the serum it should form a sharply defined layer above it. If the extract mixes 
with the serum, at the point of contact, the test will be ruined. However, if the extract 
does not mix with the serum, and the two fluids form, at the point of contact, a sharply 
defined division line, the technic has been properly executed. In case the antigen was 
prepared from anthrax material, a cloudy, grayish white ring will form immediately at 
the point of contact of the two fluids. This ring gradually increases in density for 
some time. It may be seen best in an oblique light. The test tubes used in this reac- 
tion do not necessarily have to be sterile, but it is imperative that they should be 
scrupulously clean, for if they are not clear the ring may not be seen. 
In case the antigen is prepared from material taken from an animal that did not have 
anthrax, this ring will not occur, or if it does occur will not take place under fifteen 
minutes. In case the reaction is positive, the ring remains visible for two hours, and 
longer, after which it disappears entirely. A test, however, may be read on the suc- 
ceeding day by the presence of sediment in the bottom of the tubes containing the 
positive reaction. The tubes containing negative reactions will show no precipitation. 
Control. Schutz and Pfeiler state that with reliable sera the control tests may seem 
superfluous, but still they do not wish to discard them. They make control tests only 
with suspicious material, and not with material known to be negative or positive. They 
make the statement that the extract, in every case, should be tested with normal serum 
from the same species of animal from which the antigen originates. They also use a 
positive and negative control for their antigen in routine work. In all, this makes 
three controls which are, the normal serum, the anthrax extract and the normal organ 
extract. 
