6 
Journal of Agricultural Research 
Vol. VIII. No. i 
an antigen consisting of tuberculin and tubercle bacilli in varying pro¬ 
portions. 
In the preparation of tuberculin the liquid medium contains glycerin 
in an amount varying from 5 to 7 per cent. Besredka, in preparing his 
egg medium, eliminated entirely the glycerin. In the following experi¬ 
ments the ordinary old tuberculin freed from its glycerin contents has 
been utilized. The bacillary emulsion used by different authors was pre¬ 
pared in various ways. The emulsions consisted of finely triturated 
bacteria suspended in normal or hypotonic salt solution. Alcoholic pre¬ 
cipitates of bacterial extract were also utilized (14, 35). For the 
purposes of the experiments only defatted organisms were employed, 
because it was assumed that the waxy capsule of the bacilli might pre¬ 
vent the extraction of the endotoxins; and furthermore, it was desired 
to avoid nonspecific reactions due to the presence of lipins. Therefore 
the antigen was composed essentially of concentrated tuberculin and the 
defatted organisms. At the same time comparative tests were carried 
out with other antigens. The following are especially mentioned: 
1. Emulsions of tubercle bacilli in normal or hypotonic salt solutions. 
2. Emulsions of defatted tubercle bacilli in normal or hypotonic solutions. 
3. Unheated nonconcentrated tuberculin freed from bacilli by passage through 
Berkefeld filters. 
4. Unheated nonconcentrated tuberculin freed from bacilli by passage through 
Berkefeld filters fortified with aceton insoluble lipoids, as prepared by Noguchi. The 
aceton insoluble lipoids were previously standardized against known positive syphi¬ 
litic sera to establish its titer. One unit of the antigen was added to 10 c. c. of the tuber¬ 
culin mentioned and this mixture was then used as a menstruum in the emulsification 
of the bacilli. 
5. Besredka’s antigen—(a) as received through the courtesy of Prof. Besredka, 
(b) a modification of the same, (c) a modification of the same unheated and freed from 
bacilli by passage through Berkefeld filters. 
The modified Besredka’s medium was prepared as follows: Twenty 
c. c. each of the white and the yolk of an egg were thoroughly 
beaten in an automatic egg beater, and to the whipped material a 
solution of Tiebig’s meat extract (3:1,000) in distilled water was grad¬ 
ually added while the mixture was continuously beaten. A sufficient 
meat-extract infusion was used to make up the whole to 1,000 c. c. 
The emulsion was strained through cotton and heated to the boiling 
point, then strained again, and after the addition of 0.5 per cent of 
sodium chlorid was carefully neutralized, heated again and strained, 
and neutralized if necessary. To the neutral medium sufficient normal 
sodium-hydroxid solution was added to make the medium of 0.2 alka¬ 
linity. This medium, to which neither peptone nor glycerin was added, 
was then autoclaved at 115 0 C. for 20 minutes, and after cooling was 
kept at 37 0 for 48 hours for observation as to sterility. Human and 
bovine cultures containing a 30-days’ growth of the tubercle bacilli, 
heated for 20 minutes at 115 0 and filtered, were used as antigen. This 
antigen was standardized in the usual manner. 
