104 ANTHRAX 
PREPARATION OF THE ANTIGEN 
There are three different methods by which an antigen may be prepared from the 
suspected material, namely: The boiled extract, the shake extract and the slow 
process. He also describes a method for the preparation of an antigen made from a 
culture of the anthrax organism. This antigen should be used as a control upon the 
material to be tested by this reaction. 
The Boiled Extract. This extract consists in taking a small piece of the material to 
be examined and placing it in a test tube, and adding to it four or five volumes of normal 
salt solution. The test tube is then placed in boiling water from five to fifteen minutes. 
This yields a cloudy brownish fluid which may be cleared by filtering through filter 
paper. Two or more filtrations may be necessary. 
According to Schutz and Pfeiler, this antigen may be improved by the use of carbol 
salt solution instead of normal salt solution. They state that the carbolic acid will also 
preserve the extract so that it may be kept for later use. 
The Shake Extract. This extract consists in placing a piece of tissue about the size 
of a hazelnut in a test tube to which is added about 10 ¢.c. of normal salt solution. It 
is then thoroughly shaken. This yields a dirty, reddish fluid, which is centrifuged for 
clearing purposes. It is then filtered through filter paper or asbestos. This process 
may have to be repeated several times to obtain a clear antigen. 
The Slow Process. This consists in taking a piece of suspected material about the 
size of a hazelnut and triturating in a mortar with about ten grams of dry, white sand. 
To this is added enough chloroform to completely cover it. The chloroform is allowed 
to remain for several hours, after which it is poured off. The residue should then be 
thoroughly stirred with a glass rod and enough normal salt solution added to cover it. 
This should be stirred again and the fluid filtered into a test-tube through ordinary 
filter paper. The filtrate should be colorless or slightly yellow. If it is not clear, it 
should be refiltered until it is. 
The chloroform does not have any effect upon the mixture except to precipitate the 
hemoglobin. The precipitating substance is not soluble in chloroform, and hence the 
final extract is not weakened by this process. In cases where the organs contain only 
a few bacteria, a longer extraction by the normal salt solution may be necessary. 
The Culture Extract. For this extract, a 24-hour agar culture of the anthrax organism 
is used. Five to 10 c.c. of normal salt solution is poured over the surface of the culture 
and allowed to act for two hours at room temperature. At the expiration of that time, 
the fluid is removed and filtered through ordinary filter paper. The filtrate must be 
clear, or slightly yellowish in color. 
Schutz and Pfeiler again prefer the carbol salt solution to the normal salt, as they 
think it produces a stronger antigen. 
Of the three different methods for the preparation of the antigen, Ascoli favors the 
boiled extract. On the other hand Schutz, Pfeiler and Pickens believe the slow process 
to be the best. They contend that an antigen prepared by this method contains more 
precipitinogen than by either of the other processes. 
-For the preparation of the antigen, any of the tissues of the body may be used. The 
spleen, blood, serous or hemorrhagic exudates are preferable and in the order named. 
Of the remaining organs, the liver, lungs, kidney, muscle and skin should be men- 
tioned. In case the skin is used, plenty of subcutaneous tissue should be included with 
it. According to Negroni, the presence of anthrax may be detected in imported skins 
by this method. 
