[Reprinted from The Medical News, September 6, 1890.] 
A PRELIMINARY STUDY OF THE PTOM AIN ES 
FROM THE CULTURE-LIQUIDS OF THE 
HOG-CHOLERA GERM. 1 
By E. A. V. SCHWEINITZ, Ph.D., 
CHEMICAL LABORATORY, BUREAU OF ANIMAL INDUSTRY, WASHINGTON, D. C. 
Drs. Salmon and Smith, of the Bureau of Ani- 
mal Industry, U. S. Department of Agriculture, 
were the first to demonstrate that the substances 
produced by bacteria in their growth in culture- 
media could be used for purposes of preventive 
inoculation. This they proved by a number of 
experiments upon pigeons, in 1887. They succeeded 
in producing immunity from hog-cholera by inocu- 
lating pigeons with sterilized culture-liquids of the 
hog-cholera germ. With a view to learn more of 
the products of these and other disease-germs, a 
chemical laboratory was recently attached to the 
Bureau of Animal Industry, and for a short time I 
have been engaged in studying the products in the 
culture-liquids of the hog-cholera germ. The bac- 
teriological work was done by Dr. Moore, of the 
Bureau of Animal Industry. The advice of Drs. 
Salmon and Smith has also been very valuable, and 
it was at their suggestion that the work was under- 
1 Read before the Chemical Section of the American Associa- 
tion for the Advancement of Science, August, 1890. 2 
V. SCHVVEINITZ. 
taken. Although the experiments are not com- 
plete, we think it best to make now a preliminary 
statement in regard to some of the results obtained, 
reserving for the near future a more extended account 
of the work. 
The researches of Brieger, Baginsky, Salkowski, 
Selmi, and others, have shown that the multiplica- 
tion of a number of different bacilli produces basic 
substances of an alkaloidal character, called Pto- 
maines. Some of these bodies are very poisonous, 
and a number of them have been shown to be 
identical with certain of the artificially prepared 
amines. In general, the best results have been 
obtained by allowing the germs to multiply in beef- 
bouillon, and after a number of experiments we have 
found an acid bouillon containing 2 per cent, of 
peptone to be the most satisfactory medium for the 
growth of the hog-cholera germ. Erlenmeyer flasks 
of 500 c. c. capacity were used, the mouths of the 
sterilized flasks being closed with a cotton plug. 
After inoculating the liquid with the germ the flasks 
were allowed to stand in the incubator for from two 
to three weeks, at a temperature of about 370 C. At 
the end of this time the liquid had become cloudy, 
and considerable precipitate, due to the growth of the 
germ, was found at the bottom of the flask and in 
suspension. The fluid had a decidedly alkaline 
reaction. Careful examination showed that the cul- 
ture - liquid had not become contaminated with 
foreign germs. 
In endeavoring to isolate the chemical products 
from these liquids, the methods, with some slight T H K H O G - C H O L E K A GliRM, 
3 
modifications, by which Brieger obtained such bril- 
liant results, were followed. 
The culture-liquid, after being acidified with dilute 
HC1, was evaporated on the water-bath. The re- 
sidue was ihen extracted with 98-per cent, alcohol, 
and the filtered solution treated with mercuric chlo- 
ride. A heavy crystalline precipitate was formed 
which increased upon standing. After filtration this 
precipitate was treated with water, and decomposed 
with sulphuretted hydrogen, and the mercury sul- 
phide removed by filtration. From the filtrate, after 
removal of the excess of H2S and concentration, I 
was able to isolate cadaverine, and a primary amine 
which I have not yet identified. The filtrate from 
the mercuric chloride precipitate was freed from ex- 
cess of mercury by sulphuretted hydrogen, and the 
mercury sulphide filtered off. The residue, after 
concentrating this filtrate, was extracted with abso- 
lute alcohol, the solution thus obtained showing 
the presence of a salt of an alkaloidal character. 
The reactions were as follows: 
With phosphomolybdic acid—light-yellow pre- 
cipitate. 
With bismuth-potassium iodide—red needles. 
With phosphotungstic acid—a white precipitate. 
With potassium iodide and iodine—brown-red 
precipitate. 
With platinum chloride—yellow crystalline pre- 
cipitate. 
With gold chloride—yellow-red crystalline pre- 
cipitate. 
The double salt obtained with Pt Cl4was submitted, 
after crystallization from 96-per-cent, alcohol, to a 4 
V. SCHWEINITZ. 
preliminary analysis, giving results which correspond 
to the formula C14H34N2Pt Cl6. 
The free base I have not yet succeeded in obtain- 
ing in a pure form, and will not be ready to give this 
ptomaine a name until more is learned of its consti- 
tution. The hydrochloride of this base is soluble 
in absolute alcohol, but I have obtained the salt 
only as a thick syrup, which so far will not crystal- 
lize over sulphuric acid in vacuo. 
By treating the original culture-liquids of the 
hog-cholera germ with a large excess of absolute 
alcohol a white flocculent precipitate was obtained, a 
portion of which was soluble in water, and could 
again be precipitated by alcohol. By repeated 
treatment in this manner with water and alcohol a 
small quantity of an albuminoid body containing 
C, H, N, O, and S was finally obtained. This sub- 
stance, which we will call albumose, when dried over 
sulphuric acid in vacuo, crystallized in white trans- 
lucent plates. After drying, it was still soluble in 
water, though it dissolved with more difficulty. The 
water solution gave with PtCl4 an almost insoluble 
precipitate, appearing under the microscope as 
needle-like crystals. 
The toxic effects of this albuminoid body and of 
the ptomaine were tested by subcutaneous inocula- 
tion of guinea-pigs. At the point of the inoculation 
with the albumose there was swelling and the forma- 
tion of a hard lump, which disappeared after four or 
five days. There was also a rise in the temperature 
of the animal for a few days, but in other respects 
it seemed to suffer no inconvenience. 
After subcutaneous injection of a small quantity THE HOG-CHOLERA GERM. 
5 
(j/2 c. c. or about 0.005 gramme) of the neutral 
solution of the hydrochloride of the new base before 
referred to, a rise of temperature in the animals was 
noted for a few days, and also necrosis and slight 
ulceration at the point of inoculation, otherwise the 
animals appeared well. The salt of this base, as well 
as the albumose, are, therefore, not virulent poisons. 
It may be added that special attention was given to 
keeping the solutions sterile, by means of a Pasteur 
filter, and that the absence of germs was determined 
by making plate-cultures. 
According to an article in the Berliner klinische 
Wochenschrift, March, 1890, Brieger and Frankel 
have succeeded in isolating from culture-liquids 
(containing 10 per cent, of blood-serum) of the 
diphtheria, typhoid fever, and cholera-infantum 
germs, albuminoid bodies which they call tox- 
albumose, and which are very poisonous in small 
doses. 
From culture-liquids of beef infusion with peptone 
and 2 per cent, of blood-serum, we have also obtained 
with the hog-cholera germ an albumose, having 
chemical properties similar to those described by 
Brieger, but not virulently poisonous to guinea-pigs 
when given subcutaneously, though there was con- 
siderable ulceration at the point of inoculation. 
The detailed results of the experiments in pre- 
ventive inoculation which have been conducted are 
not quite ready for publication. The statement 
may be made in advance, however, that by inocu- 
lating guinea-pigs with certain chemical compounds 
which I have prepared, the animals have been ren- 
dered immune from hog-cholera. 6 
THE HOG-CHOLERA GERM. 
Having, therefore, made considerable progress in 
the study of the culture-liquids of this hog-cholera 
germ, and also in preventive inoculation with 
chemical compounds, the results of this line of work 
are reserved for the present, and until more detailed 
results of the experiments can be given, which are 
promised for the near future.