Some Products ok the 
Tuberculosis Bacillus and the 
Treatment of Experimental 
Tuberculosis with Anti- 
toxic Serum. 
BT 
B. A. deSCHWEINITZ, Ph.D., M.D., 
Director of the Biochemical Laboratory, Bureau of Animal 
Indostry, Department of Agriculture, Washington, D. C., 
AND 
MARION DORSET, M. D., 
Assistant in the Laboratory. 
BBPBINTED FROM THE 
Neto ¥orfc jWeUfcal /Journal 
for July £4., 1897. Reprinted from the New York Medical Journal 
for July 24. 1897. 
SOME PRODUCTS OF 
THE TUBERCULOSIS BACILLUS AND THE 
TREATMENT OF EXPERIMENTAL TUBERCULOSIS 
WITH ANTITOXIC SERUM* 
By E. A. de SCHWEINITZ, Ph. D., M. D., 
DIRECTOR OF THE BIOCHEMICAL LABORATORY, BUREAU OF ANIMAL INDUSTRY, 
DEPARTMENT OF AGRICULTURE, WASHINGTON, D. C., 
and MARION DORSET, M. D., 
ASSISTANT IN THE LABORATORY. 
So much has been written in regard to the poisons of 
the tuberculosis bacillus that a review on this occasion 
would demand too much time, and we desire to refer 
only briefly to the work which is of importance in con- 
nection with those substances which we shall describe. 
Tuberculin, as is well known, is the extract of the 
tuberculosis bacilli, including the media upon which they 
are grown. From specially prepared artificial cultures 
of the tuberculosis germ, Kuhne (1) and the writer (2) 
(Bulletin No. 7, Bureau of Animal Industry) have ob- 
tained a substance corresponding to a nucleoalbumin, 
which appeared to be the fever-producing principle of 
the germ. However, many conditions in tuberculosis 
were not accounted for by this substance, and as Ma- 
fucci (3), Prudden and Hodenpyl (4), Vissman (5), and 
others had succeeded in producing tuberculous nodules 
without necrosis by the intravenous injection of dead' 
* Read before the Association of American Physicians at the Tri- 
ennial Congress of American Physicians and Surgeons, Washington, 
D. C., May 6, 1897. 
Copyright, 1897, by D. Appleton and Company. 2 
SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
either from cultures or from bodies of the bacilli them- 
selves, some substance which might be considered ac- 
countable for the coagulation necrosis of tissue which 
takes place, a necrosis which it appears is necessary for the 
progress of the disease. This problem was undertaken by 
us more than two years ago. After many fruitless attempts 
we succeeded in isolating from artificial liquid cultures 
a crystalline substance having a melting point of 161° 
bacilli, it seemed as though it should be possible to isolate, 
to 164° C., readily soluble in ether, alcohol, and water, 
which separated from these solutions in needlelike or 
prismatic crystals showing a slight yellow tint (Plate I, 
Fig. 1). They did not give the biuret reaction. The 
solution of this substance has an acid reaction to litmus, 
Fig. 1. 
is acid in taste, and is optically inactive. The crystals 
give no precipitate with silver nitrate (AgNO3), 
platinum chloride (PtCl4), or barium hydrate (Ba 
(OH)2). The analysis showed C = 50.88 per cent., H = 
6.70 per cent., 0 = 42.42 per cent., giving a formula cor- SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
3 
responding closely to C7HloO4. This is the formula of 
teraconic acid, an unsaturated acid of the fatty series. 
The culture medium upon which the bacilli were 
grown, and from which these crystals were obtained, con- 
tained potassium acid phosphate, ammonium phosphate, 
asparagin, and glycerin, the medium used and described 
by one of us (de Schweinitz) (6) some years ago for study- 
ing their products. After the growth on this media con- 
tinues for some weeks the liquid becomes light yellow 
in color, having the appearance of a pale urine, a change 
which does not take place in the uninoculated medium 
kept under the same conditions. Efforts to obtain this 
same acid from the ordinary beef-broth cultures contain- 
Fig. 2. 
ing peptone and glycerin resulted in securing minute 
amounts of the crystals only, which it was never possible 
to purify. After noting some of the other properties of 
this acid substance we came to the conclusion that the 
presence of peptone and the nitrogenous bases of the 4 
SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
meat resulted in their combination with the crystals, 
forming compounds from which the acid could not again 
be easily extracted, even after the addition of acid. Final- 
ly, a small quantity of the crystalline substance obtained 
from the artificial cultures was added to the glycerin 
peptonized beef-broth medium, but it was impossible to 
recover it again by the methods used for the first extrac- 
tion-viz., repeated precipitation with alcohol and ex- 
traction with water and ether. The ready solubility of 
this substance in water, as well as ether, probably ac- 
counts for the difficulty of obtaining it. The uninocu- 
lated medium did not yield these crystals. When dis- 
solved in water and injected into guinea-pigs the follow- 
ing effects were noted: 
I. Healthy Guinea-pigs.-No. 314. Received 0.015 
gramme crystals. Temperature at time of injection, 
102.6° F.; temperature at 11.50 a. m. (one hour after), 
100.6° F.; temperature at 1.30 p. m., 100.2° F.; tempera- 
ture at 3 p. m., 102.4° F.; temperature at 4 p. m., 102.2° 
F. During the above period the breathing was rapid, 
with an occasional rigor. 
Guinea-pig No. 422. Weight, 284 grammes. Received 
0.0095 gramme of crystals at 12.05 p. m. Temperature 
at time of injection, 99.8° F.; temperature at 2.30 p. m., 
97.4° F. On the next day there was quite a perceptible 
swelling where the injection was made. The pig was 
chloroformed at the end of twenty-four hours and showed 
considerable inflammation at the seat of injection. The 
tissues were haemorrhagic and bathed in a serous exudate. 
The muscular tissue was much disintegrated, resembling 
the appearance from the action of a caustic. 
Guinea-pig No. 511. Weight, 183 grammes. Re- 
ceived, 0.0048 gramme in half a cubic centimetre of 
water at 11.25 a. m., subcutaneously in thigh. Tem- 
perature at time of injection, 103° F.; temperature at 
12.25 p. m. (one hour after), 101.8° F.; temperature at 
1.15 p. m., 102° F.; temperature at 3.25 p. m., 100.8° 
F. Chloroformed next day. Considerable inflammation, 
with serous exudate at seat of injection. 
Guinea-pig No. 10. Received 0.0274 gramme at 10.10 
a. m. Temperature at time of injection, 99.2° F.; tem- 
perature at 10.40 a. m., 100.2° F.; temperature at 11.15 
a. m., 100.6° F.; temperature at 11.50 a. m., 100.6° F.; 
temperature at 2 p. m., 100.2° F. During above period SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
5 
this pig showed signs of restlessness, breathed rapidly, 
and shivered. 
II.- Tuberculous Guinea-pigs.-Guinea-pig No. 181. 
Received 0.017 gramme at 10.25 a. m. Temperature at 
time of injection, 102.6° F.; temperature at 11.40 a. m., 
101.4° F.; temperature at 1.50 p. m., 102.8° F.; tempera- 
ture at 2.50 p. m., 103.4°; temperature at 3.50 p. m., 103° 
F. Pig sat drawn up in cage and shivered. 
Guinea-pig No. 259 had received virulent tubercu- 
losis two weeks previous to injection of crystals. Re- 
ceived 0.0172 gramme at 10.45 a. m. Temperature at 
time of injection, 102.4° F.; temperature at 11.45 a. m., 
101.6° F.; temperature at 3.20 p. m., 101.6° F. Distinct 
rigors and rapid breathing. 
Guinea-pig No. 377, inoculated with sputum from a 
tuberculous patient some weeks before the injection of 
crystals. Received 0.023 gramme at 11.35 a. m. Tem- 
perature at time of injection, 101.2° F.; temperature at 
11.35 a. m., 100.6° F. Trembling very noticeable. 
Guinea-pig No. 11 had received attenuated and 
virulent tuberculosis culture some time before (weight, 
448 grammes). Received 0.0096 gramme at 11.25 a. m. 
Temperature at time of injection, 103° F.; temperature 
at 12.25 p. m., 100.8° F.; temperature at 1.15 p. m., 101° 
F.; temperature at 3.25 p. m., 100.8 F. 
The idea was suggested from these experiments that 
this acid, evidently a secretion of the germ, was one of 
its most powerful weapons, that by its action upon the 
tissue the cells were first destroyed so that they could sub- 
sequently be utilized by the germ as food, and in this 
way the germ protected itself from surrounding leuco- 
cytes. To test this, crystals dissolved in sterile water 
were injected by means of a hypodermic syringe directly 
into the liver tissue. At the same time the same quantity 
of water was injected into a check in the same way. 
After forty-eight hours the check and experimental ani- 
mals were killed. The check failed to show any effect, 
while the other exhibited a liver with several light spots. 
A repetition of this experiment gave the same results. 
No effort was made to recover these crystals from the 
liver as the amount used was too small. We did not test 
the effect upon the liver by an intravenous injection, 
as would otherwise have been done, because we had 6 
SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
found that there was a comhination of this acid sub- 
stance with the albuminoids or bases, and any intra- 
venous injection would have resulted in its immediate 
conversion into a modification by uniting with the albu- 
minoids in the blood. Further, the growth of the germ 
in the body is localized, and where localized the necrotic 
areas are apparent, so that the fairest test was to bring the 
substance as soon as possible in contact with the tissue. 
The experiments in injections of the animals and appear- 
ance of sections follow: 
Injection of Crystals from Artificial Cultures of Ba- 
cillus Tuberculosis into the Liver of Guinea-pigs.- 
Guinea-pig No. 409. Received 0.00178 gramme in liver 
on left side at 11.45 a. m. Temperature at time of in- 
jection, 101.6° F.; temperature at 3 p. m., 102° F. 
Chloroformed October 24, 1896, at 12 m. Liver dark, 
showing one or two small white spots and apparently a 
small inflamed spot at the point where the injection 
was made. Gall bladder was injected and seemingly in- 
flamed. 
Guinea-pig No. 412. Received 0.0037 gramme in liver 
at 1.45 p. m. Temperature at 1.45 p. m., 103° F.; tem- 
perature at 3.30 p. m., 100.4° F. Chloroformed at end 
of forty-eight hours. Gall bladder congested (not so 
much as in No. 409). Liver pale in spots, and one or two 
small white areas of apparent necrosis. The liver, hard- 
ened in HgCl2, showed on microscopic examination one 
area rather well defined where the liver cells did not 
take haematoxylon well, though nuclei stain slightly. 
Guinea-pig " C " received 0.0043 gramme crystals 
in liver. Chloroformed after six days. Pig weighed six 
hundred grammes. 
Lungs were very slightly congested. In large left lobe 
of liver there were two or three comparatively large 
areas of necrosis. These spots were on the side in which 
injection was made, and the liver appeared to show the 
track of the needle. The guinea-pig was otherwise 
healthy. 
Guinea-pig " E " received 0.0023 gramme of crystals 
in liver. Chloroformed after two days. Pig weighed 
three hundred and forty-five grammes. 
All organs appeared normal, except the stomach, 
which showed a slight inflammation in its wall on the 
side which lay next to a necrosed spot in the liver. Be- 
sides this spot there were several others of considerable SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
7 
size in the liver on the side on which the injection was 
made. The section of pig " C," the one allowed to 
live six days after injection, showed, on microscopic ex- 
amination, the following: 
Stained with haematoxylon and eosin, distinct areas of 
necrosis were noted, the most marked ones near the sur- 
face of the liver. Polynuclear leucocytes are present, 
though not in large numbers, in and around the necrotic 
areas, and there was also an increase in the connective- 
tissue cells of the liver around these same areas. Plate 
I, Fig. 2, is a drawing of the liver section showing healthy 
and necrosed areas. 
Checks on Injection of Crystals into Liver.-Guinea- 
pig No. 510. Received a fourth cubic centimetre sterile 
distilled water in liver. Chloroformed after forty-eight 
hours. Post-mortem showed all organs-liver, lungs, 
spleen, etc.-normal. 
Guinea-pig No. 387. Received half a cubic centi- 
metre of sterile distilled water in liver. Chloroformed 
after forty-eight hours. Post-mortem: All organs were 
normal, excepting one or two very small pale spots in the 
liver; no necrosis. 
Prudden (7), 1892, suggests that caseation, so con- 
stantly present in tuberculosis, is probably due to a 
specific metabolic product of the bacillus. 
It seems very reasonable to conclude from our ex- 
periments that we have here the substance formed by 
the bacillus which is responsible for the coagulation 
necrosis. 
The formula which can be deduced from the analysis 
makes this acid correspond closely to teraconic, which 
has properties very similar to those noted by us in con- 
nection with this new acid. Its identity we have not yet 
proved or disproved, as the preparation of teraconic acid 
is not completed. The amount of acid obtained is very 
small, so that we have used only a very minute portion 
of it for testing its immunizing property. A single in- 
jection of 0.0020 gramme was sufficient to keep the 
animals alive some weeks longer than the checks, and its 
solution appeared to exert some slight bactericidal influ- 
ence. 
As this substance seemed to be a temperature-reduc- 
ing principle in healthy and diseased animals, we en- 8 
SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
deavored to separate the fever-producing principle inde- 
pendently. The crystals were always found in the cul- 
ture liquid, and only minute amounts could be obtained 
for the bacilli themselves that had been grown on liquid 
medium. Accordingly, these bacilli, carefully filtered 
without heat, were washed in cold water, and next ex- 
tracted with hot water. This hot water extract contained 
an albuminoid which caused the tuberculin reaction in 
guinea-pigs and calves upon repeated injections. 
Roux and Nocard (8) state that they have a tubercu- 
lin which will give reactions almost indefinitely, but do 
not describe its method of preparation. Whether this is 
the same substance that we have obtained I am unable to 
say, but certain it is that the tuberculin prepared in 
the way we have indicated will give reactions four or five 
times in succession, where the reaction with tuberculin 
as prepared in the ordinary way fails after the second 
time. The conclusion is a fair one, I think, that the 
fever-reducing principle having been removed, to an 
extent, if not entirely, the immunity to the fever-pro- 
ducing principle is much more slowly acquired. Our 
tests upon guinea-pigs and tuberculous calves were made 
with only one day intervening between the injections 
(see Table I). 
In the Deutsche medicinische Wochenschrift for April 
1, 1897, Dr. R. Koch (9) describes some new tuberculin 
preparations. The dried tuberculosis bacilli were taken 
(the culture medium used is not mentioned), finely pow- 
dered and centrifugalized with distilled water. The 
opalescent solution obtained, tested upon animals, gave 
the tuberculin reaction. The residual germs were sub- 
mitted to this treatment a number of times, until finally 
all were practically dissolved. The latter solutions in 
large doses caused a reaction, but in small quantities did 
not produce this result, and seemed to exert both an im- 
munizing and curative action in experimental tubercu- 
losis. Koch used for this work virulent germs, and claims 
that attenuated germs do not give an active product. My 
own work was done with bacilli purposely attenuated by 
cultivation, and the results show that very active fever- SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
9 
Date. 
No. of 
animal. 
Condition. 
Weight in 
grammes. 
Substance injected. 
temperature. 
11.20 A. M. 
1.40 P. M. 
3.05 p. m. 
March 26... . 
XI 
Tuberculous guinea pig. 
423 
| c. c. tuberculin +1 c. c. serum. 
103'2° 
96'6° 
96'2° 
492 
Tuberculous guinea-pig. 
255 
| " tuberculin. 
102'8 
105'0 
104'4 
VIII 
Attenuated tuberculosis. 
443 
j " tuberculin + c. c. serum. 
101'0 
102'4 
103'8 
XIX 
Attenuated tuberculosis. 
356 
2 " cell extract=0'0040 gramme. 
102'2 
105'6 
103'4 
513 
Healthy (check). 
210 
1 " cell extract=O'0020 gramme. 
101'4 
101'6 
101'1 
10. E0 A. M. 
12.25 p. m. 
1.50 p. m. 
March 27... . 
XI 
Tuberculous guinea-pig. 
c. c. tuberculin. 
101'2° 
102'4° 
102'4° 
492 
Tuberculous guinea-pig. 
2 " cell extract=| c. c. tuberculin. 
103'0 
103'8 
105'0 
VIII 
Attenuated tuberculosis. 
£ " tuberculin. 
103'0 
103'2 
103'8 
XIX 
Attenuated tuberculosis. 
j " tuberculin. 
102'8 
103'8 
103'0 
XX 
Attenuated tuberculosis. 
| " tuberculin + | c. c. serum. 
102'4 
95'0 
95'0 
10.25 a.m. 
12.10 p. m. 
1.45 p. m. 
March 29... . 
VIII 
Attenuated tuberculosis. 
2 c. c. cell extractc. c. tuberculin. 
101'8° 
103'8° 
103'6° 
492 
Tuberculous guinea-pig. 
2 " cell extract=j c. c. tuberculin. 
102'8 
104'4 
104'6 
XIX 
Attenuated tuberculosis. 
2 " cell extract=| c. c. tuberculin. 
102'2 
105'6 
103'0 
10.45 a. m. 
12.15 p. m. 
2.10 p. M. 
March 30.... 
492 
Tuberculous guinea-pig. 
2 c c. cell c c. tuberculin. 
102'2° 
104'2° 
104'2° 
XIX 
Attenuated tuberculosis. 
J " tuberculin. 
102'2 
103'2 
103'6 
Table I.- Tests of Cell Extract, Tuberculin, and Serum. 10 
SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
producing, fever-reducing, and probably curative prin- 
ciples can be obtained from them. It hardly seemed 
justifiable to myself or others to powder dried virulent 
bacilli and have the dust floating in the air. Koch 
further refers to two fatty acids which, in conjunction 
with Proskauer (9), had been found in the bodies of the 
germs. The writers (10) of this paper published in the 
American Chemical Journal, August, 1895, a preliminary 
study of the fats of the tuberculosis bacilli, showing the 
high content of fat in the bodies of these germs, which 
accounts for the difficulty in staining them with certain 
colors, as well as their difficult absorption. 
In a later paper, Centralblatt fur Bak. u. Parasiten- 
kunde, 1896 (11), we described briefly the different acids 
obtained from the body of the germ, both high melting 
and low melting acids, but whether or not these are iden- 
tical with those observed by Koch and Proskauer we can 
not tell from the brief mention made of them. 
From our results it seems very reasonable to think 
that the necrotic acid is the fever-reducing principle, 
the albuminoid the fever-producing principle, and the 
reason the tuberculin ordinarily does not react con- 
tinuously is on account of their presence at the same 
time. At any rate, tuberculous guinea-pigs tested succes- 
sively with tuberculin showed no reaction, while with 
this albuminoid, which we will call cell extract, a reaction 
was obtained. 
The preliminary experiments, published by one of us 
in 1894 (12), upon the production of an immunity or 
resistance to tuberculosis by attenuated cultures have 
been continued and are confirmatory of the first results, 
showing the production of great resistance and in some 
cases complete immunity. A detail of two sets of these 
experiments may be given as an instance of their general 
results (Table II). 
The first effect of the injection of the attenuated 
germ was in some instances to cause a slight decrease in 
weight; sometimes a local swelling was noted at the point 
of injection, and occasionally an enlargement of the in- 
guinal glands. This disappeared after some weeks. This SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
11 
No. 
Date of inoculation and 
amount of attenuated 
germ. 
Weight, 
Oct. 84. 
Weight, 
Nov. 3. 
Condi- 
tion. 
Date of inoculation and 
amount of virulent germ. 
WEIGHTS. 
Dec. 16. 
Jan. 7. 
Jan. 19. 
Feb. 2. 
Feb. 8. 
April 8. 
April 19. 
373 
374 
Oct. 24, c. c. 
Fiftieth generation. 
12 oz. 
10 " 
14 oz. 
11 " 
0. K. 
December 9th: 373, 375, 
and 378 dead from pneu- 
14 oz. 
16 oz. 
16 oz. 
15* oz. 
16 oz. 
16 oz. 
14 oz. 
375 
376 
Fiftieth generation. 
Fiftieth generation. 
16 " 
16 " 
15 " 
14| " 
o. k. 
0. K. 
monia ; 374, 376, and 377 
each received f c. c. of 
12 " 
15 " 
15 " 
15 " 
15 " 
17* " 
18 " 
377 
378 
Check. 
1 j c. c. attenuated germ. 
13 " 
14 " 
13f " 
14 " 
0. K. 
Thin. 
vir. tuberculosis; 4th gen- 
eration from rabbit. 
12 " 
13 " 
12* " 
Dead. 
No. 
Dec. 21. 
Dec. 26. 
Dec. 21. 
Feb. 2. 
Feb. 12. 
March 8. 
March 16. 
April 6. 
April 19. 
April 21. 
II. 
All but 
All given 
13 oz. 
14 oz. 
.16 oz. 
All, including checks (X 
16 oz. 
Chloroformed. 
III. 
X and XI 
1| c. c. of 
12 " 
15j " 
14 " 
and XI), inoculated with 
17 " 
17 oz. 
19 oz. 
IV. 
(checks) 
61st gen- 
15 " 
16 " 
17 " 
| c. c. virulent germ. 
20 " 
21 " 
20 " 
V. 
received 
eration 
13 " 
15 " 
16 " 
17| " 
19 " 
19 " 
VII. 
i c. c. of 
except 
14 " 
13 " 
14 " 
16 " 
16| " 
18 " 
VIII. 
61st gen- 
checks 
15 " 
15 " 
16 " 
17 " 
16 " 
18 " 
IX. 
eration. 
(X and XI). 
12 " 
13 " 
11 " 
14 " 
15 " 
16 " 
X. 
16 oz. ch. 
16 oz. ch. 
18 OZ. f Mov. A 
17| oz. ch. 
14 oz. ch. 
12 oz. ch. 
Dead ch. 
XI. 
15| oz. ch. 
16 " " 
? C'llOC'iC.b JJACll. V. 
17| " " 
Dead. 
XII. 
16 oz. 
16 oz. 
16 oz. 
18 oz. 
19 oz. 
19 oz. 
XIII. 
14 " 
15 " 
16 " 
18 " 
17J " 
16| " 
XV. 
15 " 
16 " 
18 " 
18 " 
14 " 
XVI. 
15 " 
15 " 
17 " 
18 " 
18J " 
20 oz. 
XVII. 
12 " 
12 " 
13 " 
15 " 
15 " 
14| " 
XVIII. 
15 " 
15 " 
16 " 
16 " 
17| " 
17| " 
XIX. 
14 " 
13 " 
14 " 
13 " 
13 " 
12| " 
XX. 
15 " 
18 " 
18 " 
20 " 
19 " 
19 " 
Table II.- Two Sets of Experiments showing the Average of Results in Experiment in which the Guinea-pigs were 
Vaccinated with Attenuated Germ and then Inoculated with Virulent Germ. 12 
SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
local swelling we consider to be due to the mechanical 
action of the body of the germ on account of their high 
fat content and possible presence of a minute amount 
of the acid causing necrosis. It does not always result 
from a subcutaneous inoculation, and an apparent im- 
munity to this action is acquired by repeated injections. 
This is well shown in horses and cows submitted to treat- 
ment with the attenuated bacilli. From six to eight 
weeks after the date of the injection of the bacilli 
guinea-pigs seem to be entirely well, and are then in- 
oculated with the virulent bacilli. As can be seen from 
the chart, the checks died within six weeks from date of 
inoculation, while the others vaccinated remained well 
four months afterward. It has appeared from the many 
experiments made that if the inoculation with the viru- 
lent bacillus is made before complete recovery from the 
treatment with the attenuated bacillus, the resistance is 
considerably less. The inoculation of the animals with 
the virulent bacillus, and subsequent treatment with a 
single injection of the attenuated germ, showed that the 
latter produced a slight resistance, but no very material 
retardation of the disease. 
The production of this partial immunity or artificial 
resistance by means of the attenuated germ (12) sug- 
gested already in 1894 the availability of this same ma- 
terial for the purpose of treating animals for the pro- 
duction of a serum which would have some effect in 
curing tuberculosis. It suggested the idea, further, that 
possibly cattle could be vaccinated with this attenuated 
germ and made immune to tuberculosis. 
Experiments tried in this latter direction will be re- 
ported later. 
Two cows and one heifer were selected for the work, 
which was conducted for us by Dr. Schroeder, in charge 
of the Experiment Station of the Bureau of Animal In- 
dustry. One of these animals was originally tuberculous; 
the other two were healthy. To the tuberculous animal 
were given large doses of tuberculin until it had received 
altogether 19,407 cubic centimetres (nineteen litres and a 
half), and as much as 1,500 cubic centimetres of tubercu- SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
13 
lin at a single dose from November, 1894, to April 20, 
1897. The other animals received injections of the atten- 
uated culture, the amount injected in fifteen months be- 
ing 11,42 5 cubic centimetres and 18,100 cubic centimetres, 
respectively, and by this we mean the liquid culture in 
toto, including the bacilli thoroughly shaken in the media, 
forming an emulsion, just as taken from the incubator 
without any further treatment. At first the injections 
produced a slight reaction and occasionally a local oedema 
and abscess. After they had been continued for some 
time this effect diminished or disappeared. The serum 
of all of these animals was tested a number of times. 
Guinea-pigs were injected with the serum in quantities 
varying from one and a half to six cubic centimetres, 
and subsequently inoculated, together with the checks, 
with a tuberculosis bacillus sufficiently virulent to kill the 
checks within four or five weeks; or the pigs were in- 
oculated with the virulent bacillus and treated by sub- 
sequent injections of the serum. Without giving the 
details of the experiments we may say that the serum 
from the cow treated with tuberculin would cause in 
the pigs a slight resistance to the virulent bacilli; the 
serum of the cows treated with the attenuated bacilli 
produced more resistance on the part of the guinea-pigs, 
or prolonged their life to some extent, but not sufficient- 
ly, as compared with the quantity of material injected, 
to make the use of cow serum appear practicable. The 
cow serum, although sterile, frequently produced ab- 
scesses in the guinea-pigs. This serum we expect to test 
again when it should be more active. 
While these experiments were in progress two horses 
had been pressed into service. They were treated by 
injecting the attenuated cultures, culture fluid, and ba- 
cilli. The first injection of five cubic centimetres caused 
a decided temperature reaction, local oedema, stiffness, 
slight loss of appetite, recovery after a few days. At first 
local abscesses were formed, which healed fairly readily. 
After a time the abscess formation ceased. After eight 
months' treatment, the dose of the culture being grad- 
ually increased up to three to four hundred cubic centi- 14 
SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
metres at a time-the total amount injected in fifteen 
months being four thousand four hundred and fifty-nine 
cubic centimetres-the serum was used for testing. It 
separated out clear and well. Two sets of illustrations 
may be given to show its action on tuberculous animals. 
In one set (Table III) the checks and two treated pigs 
died; the other two treated pigs are alive, and in per- 
fect health, apparently, after a number of months. In 
another set the checks, four in number, died within four 
to five weeks, while the treated ones lived two to three 
weeks longer, showing, on autopsy, much less disease in 
the lungs than the checks. We endeavored further to 
isolate from the serum antitoxic substances by a slight 
modification of the Brieger-Boer method. We finally 
succeeded in obtaining a small quantity of a grayish pow- 
der giving the biuret reaction, with difficulty soluble in 
water, which was used for treating guinea-pigs in the 
same way as the serum. The result was about the same 
as in the first instance. The pigs, half a pound in weight, 
were inoculated with a virulent germ and treated by a 
single injection of 0.008 gramme of this solid substance. 
They lived three or four weeks longer than the checks, 
the lungs again showing considerably less disease, and less 
necrosis was noted in the liver (Table IV). 
The effect of the serum was also tried in preventing 
the rise of temperature in tuberculous guinea-pigs and in 
saving them from a fatal dose of tuberculin. As can be 
seen from the temperature reactions in Table I, the injec- 
tions of one fourth of a cubic centimetre of diluted tu- 
berculin, and at the same time of half a cubic centimetre 
of the serum, either caused a decided reduction of the 
temperature or prevented a characteristic tuberculin re- 
action in animals weighing five hundred grammes. This 
is one way of gauging the serum. 
The result of all this work leads us to the conclusions 
that the injection of the live culture produces substances 
antitoxic to the disease which will cure tuberculous ani- 
mals; that the quantity of this substance can be increased 
gradually; that the treatment of tuberculosis is and will be 
for some time still in the experimental stage. One point, SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
15 
No. 
Weight. 
Date and amount of viru- 
lent culture. 
DATES AND AMOUNT OK SEBUM INJECTED. 
Nov. 6. 
Nov. 17. 
Nov. 25. 
Dec. 3. 
Dec. 8. 
April 19. 
434 ch. 
435 
436 
437 
438 
439 
10 oz. 
9 " 
11 " 
14 " 
9 " 
8 " 
Oct. 24th; | c. c. of viru- 
lent tubercular culture 
given to all; all ex- 
cept check received 1£ 
c. c. of serum. 
10 OZ. 
9 " +1| c. c. 
10 " +1| " 
13 " +li " 
8 " + H " 
8 " +1| " 
8| cz. 
8 " + 1| c. c. 
9 " +1| " 
11 " 4-l| " 
8 " + H " 
7 " +H " 
8 oz. 
8 " +1| c.c. 
7 " +1| " 
10 " +U " 
6 " 4-lj " 
5 " +1| " 
Dead. 
Dead. 
Dead. 
9 oz. 
10 " 
20 oz. 
20 " 
Alive and well. 
Alive and well. 
No. 
Weight. 
Date. 
Material for inoculation. 
Date. 
Weight. 
Date. 
Date. 
Weight. 
Date. 
464 
476 
478 
479 
481 
482 
484 
Check, 11 oz. 
12 " 
Check, 8| " 
Check, 8 " 
Check, 10 " 
13 " 
12 " 
Feb. 4. 
" 4. 
" 4. 
" 20. 
" 20. 
" 20. 
" 20. 
| c. c. virulent culture. 
-J- c. c. virulent culture + 
0 • 008 grs. antitoxine. 
c. c. virulent germ. 
| c. c. tuberc. virulent. 
| c. c. tuberc. virulent. 
c. c. tuberc. virulent + 
0'008 grs. antitoxine. 
c. c. tuberc. virulent + 
0 ' 008 grs. antitoxine. 
Feb. 20. 
" 20. 
" 20. 
11 oz. 
12 " 
8 " 
March 6. 
" 6. 
" 6. 
" 6. 
" 6. 
" 6. 
10 oz. 
10 " 
7 " 
9 " 
12| " 
12 " 
March 8, dead. 
March 16, dead; less disease 
than others. 
March 8, dead ; generalized 
tuberculosis. 
March 12, dead. 
April 7, dead; less disease. 
April 2, dead; less disease 
than others. 
Feb. 26. 
Dead; tu- 
berculosis. 
Table III.-Serum from Horse Injected with Attenuated Culture used on Tuberculous Guinea-pigs. 
Table IV.- Tests of Dry Antitoxic Material from Serum from Vaccinated Horse. 16 
SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
however, must be remembered-viz., that while it may 
be difficult to cure the disease in a guinea-pig, where its 
course is very rapid-a virulent bacillus requiring only 
from four to five weeks to kill-it might be much easier to 
check the disease when more prolonged in action, as in 
the majority of cases in man. Again, in addition to some 
form of specific treatment for the disease, man usually 
has the advantage of being placed under the best possible 
surroundings as to diet, climate, .etc., and every effort is 
made to aid the improvement of the patient, while with 
experimental animals the conditions are different. 
The experimental results obtained lead undoubtedly 
to the conclusion that while the treatment with anti- 
toxic serum is still in the experimental stage and should 
be as yet only used in sanitariums and under the best 
conditions, we are on the road to success in the treatment 
of this disease and nearer our goal than ever before. 
In an experimental way the antitoxic serum as prepared 
in our laboratory has been used by Dr. Stubbert at the 
Loomis Sanitarium and some by Dr. Trudeau at Saranac 
Lake, as well as by Dr. C. W. Richardson in this city 
(Washington, D. C.). 
Dr. Stubbert, out of six cases treated, reports one 
cured and others improved. Dr. C. W. Richardson notes 
decided improvement in his cases, while Dr. Trudeau, 
who has used some of the serum for a short time only, 
noted in one case a reduction of temperature which may 
have been due to this serum. 
Maragliano, Babes, Behring, and Paquin are the other 
principal workers in the preparation of an antitoxic 
serum for tuberculosis. 
Maragliano (13) gives the method he has used for the 
production of antitoxic serum, and notes that there is 
present in the cold filtered cultures of the tuberculosis 
bacilli, a substance which causes the reduction of tempera- 
ture, and another not destroyed by heat, which causes the 
rise of temperature. In all probability, without isolating 
the principle, Maragliano was using solutions of the 
crystalline substance we have described in the beginning 
of this paper. While this is not destroyed by heat, as SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
17 
he seems to think, it does undergo some change by com- 
bining probably with the albuminoid matter in the 
media, and thus losing its distinct property as a tem- 
perature-reducing substance. Or, more probably, its tem- 
perature-reducing property is disguised by the presence 
of the temperature-producing principle extracted by hot 
water. The serum which he claims to obtain from treat- 
ment of the animals is said to have some effect in re- 
ducing the temperature and apparently improving the 
disease. 
Babes (14), reviewing a portion of the work upon the 
treatment of tuberculosis with serum, comes to the con- 
clusion that he is the first individual to have discovered 
any antitoxic properties in the serum from treated ani- 
mals; that there is an antitoxic substance present in 
this serum, but that it has not yet been brought to a 
sufficient development to warrant general use. 
Our experiments lead us to conclude that while the 
injections with tuberculin produce a serum containing 
antitoxic material, the amount of this is small, and that 
the injection of the live culture is the proper treatment. 
We can not agree to the statements made that horses 
are unsuitable for the work. Mules and donkeys may 
perhaps give results more quickly, but horses seem to be 
eminently satisfactory. At no time have we found that 
the horse serum produces toxic effects, although this 
has been noted in the cow serum. 
If the antitoxic serum treatment and other methods 
for tuberculosis could be freed for the present from their 
commercial aspect, and careful, systematic experiments 
continuously conducted in numerous hospitals and sani- 
tariums, this or a similar modified method of treatment 
could be looked to for good results. 
When tuberculosis can be uniformly cured in guinea- 
pigs as certainly as diphtheria, then does the commercial 
aspect become a fair and legitimate one. 
Articles referred to in this Paper. 
1. Kiihne. Zeit. f. Biologic, Bd. xxx, 1894, p. 221. 
2. De Schweinitz. Bulletin No. 7, Bureau of Ani- 
mal Industry, 1894. 18 
SOME PRODUCTS OF TUBERCULOSIS BACILLUS. 
3. Mafucci. Centralb. fur allg. Path., Dec. 15,1893. 
4. Prudden and Hodenpy]. New York Med. Jour- 
nal, June 6 and 20, 1891. 
5. Vissman. Archiv fur path. Anat. u. Phys, und 
fur klin. Med., 1892, cxxix, p. 163. 
6. De Schweinitz. New York Med. Journal, March 
11,1892. 
7. Prudden. New York Med. Journal, September 10, 
1893. 
8. Roux and Nocard. Receuil de m&d., 1897. 
9. Koch and Proskauer. Deut. med. Woch., April 1, 
1897. 
10. De Schweinitz and Dorset. Journal of the Amer. 
Chem. Society, August, 1895.-Bulletin No. 13, 1896, 
Bureau of Animal Industry. 
11. De Schweinitz and Dorset. Centralb. f. Bak. u. 
Parasit., vol. xix, 18, 19, 1896. 
12. De Schweinitz. Med. News, December 8, 1894. 
13. Maragliano. Revue de la tuberculose, juillet, 1896. 
14. Babes. Zeit. f. Hygiene, Bd. xxiii, lift. 3. 
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