THE TOXINES PRODUCED BY THE STAPHYLO- 
COCCUS PYOGENES AUREUS. 
BY 
S. P. KRAMER, M.D., 
OF CINCINNATI, OHIO. 
FROM 
THE MEDICAL NEWS, 
November 12, 1892. [ Reprinted from The Medical News, November 12, 1892.] 
THE TOXINES PRODUCED BY THE STAPHYLO- 
COCCUS PYOGENES AUREUS. 
By S. P. KRAMER, M.D., 
OF CINCINNATI, OHIO. 
The following is a report of the results of an 
investigation of the metabolic products formed by 
the staphylococcus pyogenes aureus carried on in 
the Biological Laboratory of the University of Cin- 
cinnati. 
Two cultures were used ; one kindly furnished me 
by Dr. Novy, of Ann Arbor, recently obtained from 
the blood of a child that had died of pyemia; the 
other a culture of two years' standing. No appre- 
ciable difference in result was recognized. A 
bouillon containing i per cent, of meat extract 
and i per cent, of peptone was used as a culture- 
medium. The germs were grown in Erlenmeyer 
flasks containing from 40 to 50 c.c. After remain- 
ing in the incubator at a temperature of from 350 
to 370 C. for seven days, the cultures were passed 
through Chamberland filters and the sterility of the 
filtrate established both by microscopic examination 
and inoculation upon agar. 
This filtrate injected into rabbits produced no 
apparent symptoms. Thus, a rabbit weighing two 
pounds received 8 c.c. without effect. A daily 2 
injection of 2 c.c, during a period of seven days 
gave a similar result. This result was to be ex- 
pected in view of the well-known resistance of the 
rabbit to this microorganism. The injection of 
four centigrams of the filtrate in the left forearm 
of the author was followed by marked local and 
general reaction. Five hours after the injection the 
temperature had risen from 98.4° to 99.8° F., pulse 
96, accompanied by headache, pain in the back, 
flushing of the face,and general malaise. Locally,the 
point of injection was red, tender, and showed slight 
edema. Within the next eight hours the tempera- 
ture returned to normal, some headache and malaise 
remaining during the succeeding twelve hours. The 
local inflammation increased during this time, but 
subsided completely after seventy-two hours, and 
was followed by slight desquamation. Another in- 
jection of 6 c.c. was followed by essentially the 
same symptoms, though to a less degree, a rise 
of temperature from 98.5° to 990, pulse 90, slight 
headache, and a marked local inflammation. 
From these experiments the conclusion was 
reached that the staphylococcus produced in its 
culture one or more chemical compounds which, 
when injected, gave rise to characteristic general 
symptoms and marked local irritation. 
The next step in the investigation was the de- 
termination of the properties of these compounds, 
and, if possible, their location. When subjected to 
a temperature of 6o° C. for half an hour the filtrate 
produced no general symptoms, even when large 
doses were injected. The local effect, however, 
remained unchanged. A quantity of filtrate was 3 
added to ten times its volume of absolute alcohol. 
A grayish-white precipitate resulted. This was 
allowed to settle to the bottom, the supernatant 
alcohol drawn off, and the precipitate repeatedly 
washed with 90 per cent, alcohol. After purifica- 
tion in this way the precipitate was dried in a 
vacuum at 330 C., and a light tan-colored powder 
was obtained. 
This powder proved to be only partly soluble in 
water, but completely soluble in a 1 per cent, solu- 
tion of sodium carbonate. A portion of this powder 
was then rubbed up with water and the insoluble 
portion separated by filtration. The filtrate was 
then thrown into ten volumes of alcohol; the result- 
ing precipitate was re-dissolved and again precipi- 
tated with alcohol. After repeating this operation 
six times, and drying in a vacuum, a light grayish- 
white powder was obtained, albuminous in charac- 
ter, which, when injected, produced a very slight 
local and no general reaction. Another portion of 
the original powder was dissolved in a 1 per cent, 
solution of sodium carbonate. The injection of 
this in very small doses, i. e. (1 mg.) produced a 
very marked local irritation. 
In all these experiments control-injections of 
bouillon were made, with negative results. 
The insoluble alcoholic precipitate gave the fol- 
lowing chemical reactions: 
The light, tan colored powder was incompletely 
soluble in water and dilute saline solutions. When 
water was added the powder became a seal-brown 
flocculent mass. It was completely soluble in a 1 4 
per cent, solution of sodium carbonate; insoluble 
in alcohol and ether. 
It was precipitated from its solution by alcohol, 
mercuric chloride, ammonium sulphate, and acetic 
acid. 
It gave the biuret reaction, and with Millon's 
reagent behaved like a proteid. 
It was not precipitated by heat, nitric acid and 
heat, sodium chloride, or sodium sulphate. 
By virtue of these reactions the body is to be classed 
among the albumins. To which particular variety 
it belongs is not at present apparent. 
As the result of this investigation the following 
conclusions may be drawn : 
The staphylococcus pyogenes aureus produces 
toxic albuminous products of two kinds: 
i. A readily diffusible substance, difficult of 
isolation, very susceptible to heat and chemical 
reagents, which, when absorbed in the human sys- 
tem, produces symptoms of general disturbance. 
2. A less diffusible albuminous substance, not 
very susceptible to a temperature of from 6o° to 700 
C., or to chemical reagents, which, when injected 
in the human subject, produces at the point of injec- 
tion a well-marked inflammation. 
Christmas, working in Pasteur's laboratory, was 
the first investigator to take up this point. He 
obtained from bouillon-cultures of the staphylococ- 
cus pyogenes aureus, by the addition of alcohol, a 
precipitate that produced suppuration when injected 
in the anterior chamber of the eye and subcutaneous 
tissue of rabbits. 
In the same way Brieger and Fraenkel obtained a 5 
toxalbumin insoluble in water, which proved fatal 
when injected in rabbits or guinea-pigs. On post- 
mortem examination of such animals they found the 
following changes: Intense inflammation at the 
point of injection, with swelling and reddening of 
the neighboring tissues; at times a purulent necro- 
sis at the point of injection, which in some cases 
extended through the abdominal wall and gave rise 
to an antiseptic purulent peritonitis. The amount 
of toxalbumin injected by these authors is not 
stated, but it must have been a very large quantity 
in order to produce such grave changes. I should 
judge that in these experiments the animals were 
killed by the violence of the local insult rather than 
by the absorption of any general toxic product. 
My investigation would lead me to believe that 
the rabbit is proof against the general toxine pro- 
duced by this germ, and that, moreover, the chemi- 
cal operations employed in endeavoring to isolate 
it would destroy any toxic properties that it may 
have possessed. 
There is one other point to which I desire to call 
attention-the therapeutic possibilities offered by 
these substances. If we take the sterile filtrate of 
staphylococcus cultures, and heat it for half an hour 
at 6o° C., we have an irritant that I think will prove 
of considerable value. We have a counter-irritant 
that, when injected in very small doses, produces a 
well-marked irritation; in larger doses, almost a true 
erysipelatous inflammation. There are no dangers 
of general disturbance attending its use, there is no 
abrasion of the surface to become infected, and the 
effect to be produced is thoroughly within control. 6 
If a y per cent, of carbolic acid be added to it 
the fluid will keep indefinitely, or, better still, a i 
per cent, solution of the alcoholic precipitate in i 
per cent, sodium carbonate solution could be used. 
Such solutions would be of great value, both as 
counter-irritants and as a means of hastening sup- 
puration in furuncles, etc. 
If, on the results of these investigations, it is per- 
missible to construct a theory of suppuration as 
produced by the staphylococcus, it would be briefly 
as follows: The staphylococcus, during its growth 
in the tissues, produces a toxine that, when absorbed, 
gives rise to the general symptoms attending a local 
suppurative process. Furthermore, it produces a 
toxic substance that is highly irritant, and that pos- 
sesses a strongly positive chemotaxis for the leuko- 
cytes. The duty of the local leukocytosis is two- 
fold : First, by virtue of their phagocytic properties 
the leukocytes restrain the growth of the germ, and 
secondly, the local necrosis caused by their effusion 
enables the organism to discharge the bacteria, toxic 
products, and necrotic tissue present. The Medical News. 
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