Improvements in the Manufacture of Remedial Serum.
Year: 1896

page_text: Date of Application, 28th Jan., 1896
Complate Concification Loft 20th Out 1006
Accented
12th Doo 4906
Improvements in the Manufacture of Remedial Serum.
A communication from abroad by FRANZ NIEMANN, of Bâle, Switzerland,
Bacteriologist.
I, GEORGE BELOE ELLIS of the Firm of J. C. Mewburn and Ellis of 55 and
56 Chancery Lane in the County of London, Chartered Patent Agents, do hereby
declare the nature of this invention to be as follows:-
This invention has reference to the manufacture of remedial serum and more
particularly of serum for the cure or retardation of the progress of tuberculosis.
Since the discovery of tuberculin by Koch frequent attempts have been made to
prepare a tuberculin-antitoxine for remedial purposes.
Amongst the earlier attempts, injections of pure living cultivations of tubercle
bacilli were made into horses and other animals, and blood drawn from the tuberculous
animals after several weeks. The serum separated from this blood was stated to
possess a slight specific anti-toxic action on tuberculous subjects, but the animal
serving for the preparation of the serum almost invariably died before any con-
siderable quantity of anti-toxine was elaborated in its system, and consequently
experiments in this direction were too few for any definite result to be arrived at.
In place of tubercle bacilli, injections were then tried of tuberculin, which is a
sterilized glycerine extract of pure cultures of the tubercle bacillus. Boinet and
E. Managliano have stated that it is possible by repeated injections with tuberculin, of
which no quantities are given, to obtain a serum from the inoculated animal capable
of exerting a remedial effect on tuberculous subjects. It appears, however, from their
reports, that only a brief temporary delay in the progress of the disease was afforded
by the use of this serum, and Managliano himself admits that its injection is
objectionable in advanced stages of consumption or where the infected area is
extensive, whilst in similar cases Boinet observed as sequelae of treatment with his
serum fever, hemoptoea, night sweats and throat complications.
The employment of tuberculin alone for injection therefore appears as futile as the
employment of tubercle bacilli in the manufacture of antitoxic serum of remedial
value.
After careful consideration of the probable causes of failure and studying the
disadvantages, both practical and theoretical, of the above methods, my corre
spondent succeeded, after many trials, in discovering a procedure different from
the foregoing which enables tuberculous remedial serum to be prepared of high
anti-toxic value. Especially was he led to his conclusions by the consideration that
glycerine is a blood poison, and when introduced into the circulation partially dissolves
or shrivels up the red corpuscles and causes disturbances and stoppages in the blood
vessels, especially in the neighbourhood of the pulmonary organs, which set up
conditions promoting parenchymatous change. It therefore appeared to him that only
very small quantities of tuberculin could be injected without running the risk of
developing deleterious bye-effects from glycerine poisoning.
For the preparation of remedial serum according to the new method, it is preferred
to employ one- to two-year old goats, though other animals, such as horses, asses,
mules etc. may be employed should it be found impracticable to manufacture
sufficient quantities of serum from the smaller animals. In the following directions
for effecting immunity the quantities therefore only refer to healthy goats 24 kilos. in

page_text: weight, but the proportions of materials used to the animal weight remain substan-
tially identical when larger or smaller animals are employed.
The healthiness of the animal should first be ascertained by keeping it under close
observation for about four weeks prior to the commencement of the treatment, during
which period the weight of the animal should not decrease but rather increase.
In carrying out this invention the treatment to which the animals are subjected in
order to procure the desired degree of immunity and the elaboration of a sufficient
quantity of tuberculin-antitoxine in their systems to render the preparation of my
remedial serum possible, should be effected in three distinct stages, which I term the
preliminary treatment, the special treatment and the final treatment, respectively.
PRELIMINARY TREATMENT.
A subcutaneous injection of e.e. tuberculin is made preferably in the neck of the
animal hy means of a sterilized Pravatz syringe or other suitable instrument. The
tuberculin which I employ for this purpose is preferably made in the following manner:
A pure cultivation of tubercle bacilli fron tuberculous human organs is made in
glycerine-goat-blood-serum, and then a sterilized bouillon containing 12 grams peptone,
6 grains sodium chloride, 15 grams glycerine and 100 c.c. sterilized goat-blood-serum
per 1000 e.c. of bouillon is inoculated therewith. The inoculated bouillon is then kept
in an incubator for eight weeks at 41° (. If at the end of that period a thick crust
of tubercle bacilli has formed upon the surface of the bouillon and microscopical
examination reveals the complete purity of the cultivation, the culture is sterilized
by heating for 2 hours at 100° C. and then concentrated to one-tenth of its bulk,
filtered through a Chamberland filter, and the filtrate evaporated to one-half. The
dark-yellow thick liquid so obtained is the tuberculin employed in the preliminary
treatment.
After the first injection of 2 c.c. of this tuberculin there is usually a rise in
temperature in goats of 1° to 14° C.; after 48 hours a second injection of 4 c.c.
tuberculin is made, and if there is no subsequent rise, in temperature within 24 hours,
at the end of 48 hours after the second injection, a tbird injection of 4 c.c. tuberculin
is made. If, on the other hand, a reaction occurs after 24 hours, the third injection
is unnecessary.
The preliminary treatment is now complete, and I proceed to the special treatment
for which I employ the precipitated active component or principle of tuberculin in
order to avoid the danger of, more particularly, glycerine poisoning.
SPECIAL TREATMENT.
The active principle of tuberculin for injection at this stage is prepared free from
glycerine preferably in the following manner: 500 c.e. tuberculin is heated to 80° C,
then introduced into a high cylindrical vessel of about 1500 c.c. capacity and
700 c.c. 90 per cent. alcohol containing (8 per cent. carbolic acid added, constantly
stirring. A light voluminous precipitate is formed almost immediately which is
induced to deposit itself by centrifugating for a quarter of an hour; the supernatant
liquid is then poured off from the dark-brown deposit, and the latter digested for
12 hours at 40° C. with 60 per cent. alcohol containing 15 per cent. ammonium
sulphate in solution. To ensure complete precipitation it is then allowed to stand a
further 12 hours at ordinary temperature, whereupon the alcohol is carefully poured
off and the precipitate brought upon a filter, where it is washed with a mixture
consisting of 3 parts ethyl alcohol, 1 part benzene, 1 part chloroform, and 0-5 part
amyl alehol and finally dried in an exsiccator over sulphuric acid.
The preparation thus obtained consists of a yellowish-white powder, which dissolves
fairly easily in water. 7 milligrammes of this preparation equals 1 c.c. tuberculin in
activity. As it commences to decompose after 6 to 8 days when dissolved in water,
only the quantity required for immediate use should be dissolved at one time.
The animal already subjected to the preliminary treatment receives in aqueous

page_text: solution 15 milligrammes of the preparation as a first injection, and 25 milligrammes
as a second, which is injected after 48 hours if no rise in temperature has meanwhile
taken place. If a rise in temperature takes place, at least 24 hours must be allowed
to elapse after the temperature has fallen before further treatment is proceeded with.
In the early stages of the treatment the animals exhibit great sensitiveness and react
almost regularly with slight fever after each injection. It is therefore necessary to
raise the dose gradually, only 10 milligrammes at a time. But after the animals have
been accustomed to receive 50 milligrammes at a single injection without a marked
general disturbance of health, it is possible to progress more quickly. From
50 milligrammes the dose may be raised to 75, then successively to 100 and
125 milligrammes, but a pause of 3 or 4 days is necessary between each injection.
Naturally it is impossible to draw hard and fast rules in this case, as each animal
possesses an individuality of its own which requires individual attention. The special
treatment can be regarded as complete! when for each kilo bo ly weight of the animal
2 grammes of the above described cone ntrated preparation free from glycerine have
been injected.
FINAL TREATMENT.
For final treatment I employ a preparation for injection which is preferably
produced in the following manner: Bouillon of the composition above described is
inoculated with tubercle bacilli under the precautions defined above and allowed to
stand in the incubator at 40° C. for 2 weeks. After this period has elapsed the
bouillon is boiled for 2 hours over wire gauze, brought into an agate mortar and
the whole well triturated. Of this sterilized liquid containing the dead remains of
the bacilli, 7 to 10 e.c. is injected into each animal according to its weight. In
consequence of this inoculation local suppuration and abscesses are always formed,
but are quickly healed under antiseptic treatment.
The animals having undergone the above three treatments in succession are now
kept preferably about 3 or 4 weeks before drawing blood from them for the purpose
of preparing the remedial serum. Then by means of a trocar 05 to 0.75 litres blood
is drawn from the jugular vein under aseptic precautions, and after congulation the
serum is poured off, filtered through a Chamberland filter, 04 per rent. carbolic
acid added, and filled into sterilized bottles. It is then ready for therapeutical
employment.
Experiments on animals have proved that this remedial serum, prepared as above,
possesses specific antitoxic properties, which cause it to heal light cases of tuberculosis
and to stay the progress of severe and advanced cases of the disease.
Dated this 28th day of January 1896.
J. C. MEWBURN & ELLIS,
55 and 56 Chancery Lane, London, Chartered Patent Agents.
ETE SPE
Improvements in the Manufacture of Remedial Serum.
A communication from abroad by FRANZ NIEMANN, of Bale, Switzerland,
Bacteriologist.
I, GEORGE BELOE ELLIS of the Firm of J. . Mewburn and Ellis of 35 and
56 Chancery Lane in the County of London, Chartered Patent Agents, do hereby
declare the nature of this invention and in what manner the same is to be performed, to
be particularly described and ascertained in and by the following statement :-
This invention has reference to the manufacture of remedial serum and more
particularly of serum for the cure or retardation of the progress of tuberculosis.

page_text: Since the discovery of tuberculin by Koch frequent attempts have been made to
prepare a tuberculin antitoxine for remedial purpo-es.
Amongst the earliest attempts, injections of pure living cultivations of tubercle
bacilli were made into horses and other animals, and blood drawn from the tuber-
culous animals after several weeks. The serum separated from this blood was stated
to possess a slight specific anti-toxic action on tuberculous subjects, but the animal
serving for the preparation of the serum almost invariably die before any
considerable quantity of anti-toxine was elaborate in its system, and consequently
experiments in this direction were too few for any definite resalt to he arrived at.
In place of tubercle bacilli, injections were then tried of tuberculin, which is a
sterilized glycerine extract of pure cultures of the tubercle heillus. Boinet and
E. Managliano have stated that it is possible by repeated injections with tuberculin,
of which no quantities are given, to obtain a serum from the inoculated animal
capable of exerting a remedial effect on tuberculous subjects. It appears however,
from their reports, that only a brief temporary delay in the progress of the dise.ise
was afforded by the use of this serum, and Managliano himself admits that its
injection is objectionable in advanced stages of consumption or where the infected
area is extensive, whilst in similar cases Boinet observed as sequelae of treatment
with his serum fever, hemoptea, night sweats and threat complications.
The employment of tuberculin alone for injection therefore appears as futile as
the employment of tubercle bacilli in the manufacture of antitoxic serum of
remedial value.
After careful consideration of the probable causes of failure and stu lying the
disadvantages, both practical and theoretical, of the above methods, my correspondent
succeeded after many trials, in discovering a procedure different from the foregoing
which enables tuberculous remedial serum to be prepared of high autitoxic value.
Especially was he led to his conclusions by the consideration that glycerine is a
blood poison, and when introduced into the circulation partially dissolves or shrivels
up the red corpuscles and causes disturbances and stoppages in the blood vessels,
especially in the neighbourhood of the pulmonary orgaus, which set up conditions
promoting parenchymatous change. It therefore appeared to him that only very
small quantities of tuberculin could be injected without running the risk of
developing deleterious bye-effects from glycerine poisoning.
For the preparation of remedial serum according to the new method, it is
preferred to employ one to two-year old goats, though other animals, such as
horses, asses, mules &c. may be employed should it be found impracticable to
manufacture sufficient quantities of serum from the smaller animals. In the
following directions for effecting immunity the quantities therefore only refer to
healthy goats 24 kilos. in weight, but the proportions of materials used to the
animal weight remain substantially identical wlien larger or smaller animals are
employed.
The healthiness of the animal should first be ascertained by keeping it under
close observation for about four weeks prior to the commencement of the treatment,
during which period the weight of the animal should not decrease but rather
increase.
In carrying out this invention the treatment to which the animals are subjected
in order to procure the desired degree of immunity and the claboration of a
sufficient quantity of tuberculin-antitoxine in their systems to render the preparation
of my remedial serum possible, should be effected in three distinct stages, which
I term the preliminary treatment, the special treatment and the final treatment,
respectively.
PRELIMINARY TREATMENT.

page_text: manner-A pure cultivation of tubercle bacilli from tuberculous human organs is
made in glycerine-goat-blood-serum, and then a sterilized bouillon containing
12 grams peptone, 6 grams sodium chloride 15 grams glycerine and 100 c.c.
sterilized goat-blood-serum per 1000 c.c. of bouillon is inoculated therewith. The
inoculated buillon is then kept in an incubator for eight weeks at 41° C. If at the
end of that period a thick crust of tubercle bacilli has formed upon the surface
of the bouillon and microscopical examination reveals the complete purity of the
cultivation, the culture is sterilized by heating for 2 hours at 100° C. and then
concentrated to one-tenth of its bulk, filtered through a Chainberland filter, and the
filtrate evaporated to one-half. The dark yellow thick liquid so obtained is the
tuberculin employed in the preliminary treatinent.
After the first injection of 2 c.c. of this tuberculin there is usually a rise in
temperature in goats of 1° to 14° C.; after 48 hours a second injection of 4 c.c.
tuberculin is made, and if there is no subsequent rise in temperature within 24 hours,
at the end of 48 hours after the second injection, a third injection of 4 c.c. tuberculin
is made. If, on the other hand, a reaction occurs after 24 hours, the third injection
is unnecessary.
The preliminary treatment is now complete, and I proceed (after an interval of
from eight to ten days) to the special treatment for which I employ the precipitated
active component or principle of tuberculin in order to avoid the danger of more
The active principle of tuberculin for injection at this stage is prepared free from
glycerine preferably in the following manner :-500 c.c. tuberculin is heated to 80° C.,
then introduced into a high cylindrical vessel of about 1500 c.c. capacity and 700 c.c.
90 per cent. alcohol containing 0.8 per cent. carbolic acid added, constantly stirring.
A light voluminous precipitate is formed almost immediately which is induced to
deposit itself by centrifugating for a quarter of an hour; the supernatant liquid is
then poured off from the dark-brown deposit, and the latter digested for 12 hours at
40° C. with 60 per cent. alcohol containing some neutral mineral salt, preferably
1.5 per cent. ammonium sulphate in solution. To ensure complete precipitation it is
then allowed to stand a further 12 hours at ordinary temperature, whereupon the
alcohol is carefully poured off and the precipitate brought upon a filter, where it is
washed with a mixture consisting of 3 parts ethyl alcohol, 1 part benzene, 1 part
chloroform, and 0-5 part amyl alcohol and finally dried in an exsiccator over
sulphuric acid.
The preparation thus obtained consists of a yellowish-white powder, which dissolves
fairly easily in water. 7 milligrammes of this preparation equals 1 c.c. tuberculin in
activity. As it commences to decompose after 6 to 8 days when dissolved in water,
only the quantity required for immediate use should be dissolved at one time.
The animal already subjected to the preliminary treatment receives in aqueous
solution 15 milligrammes of the preparation as a first injection, and 25 milligrammes
as a second, which is injected after 48 hours if no rise in temperature has meanwhile
taken place. If a rise in temperature takes place, at least 24 hours must be allowed to
elapse after the temperature has fallen before further treatment is proceeded with.
In the early stages of the treatment the animals exhibit great sensitiveness and react
almost regularly with slight fever after each injection. It is therefore necessary to
raise the dose gradually, only 10 milligrammes at a time. But after the animals
have been accustomed to receive 50 milligrammes at a single injection without a
marked general disturbance of health, it is possible to progress more quickly. From
50 milligrammes the dose may be raised to 75, then successively to 100 and 125 milli-
grammes, but a pause of 3 or 4 days is necessary between each injection. Naturally
it is impossible to draw hard and fast rules in this case, as each animal possesses an
individuality of its own which requires individual attention. The special treatment

page_text: FINAL TREATMENT.
For final treatment 1 employ a preparation for injection which is preferably
produced in the following manner: Bouillon of the composition used in the preliminary
treatment is inoculated with tubercle bacilli under the precautions defined above and
allowed to stand in the incubator at 10° C. for 2 weeks. After this period has
elapsed the bouillon is boiled for 2 hours over wire gauze, brought into an agate
mortar and the whole well tritnrated. Of this sterilize liquid containing the dead
remains of the bacilli, 7 to 10 c.c. is injected into each animal according to its weight.
In consequence of this inoculation local suppuration and abscesses are always formed,
but are quickly healed under antiseptic treatment.
The animals having undergone the above three treatments in succession are now
kept preferably about 3 or 4 weeks before drawing blood from them for the purpose
of preparing the remedial serum. Then by means of a trocar 05 to 0-75 litres
blood is drawn from the jugular vein under aseptic precautions, and after coagulation
the serum is poured off, filtered through a Chamberland filter (4 per cent. carbolic
acid added, and filled into sterilized bottles. It is then ready for therapeutical
employment.
Experiments on animals have proved that this remedial serum, prepared as above,
possesses specific antitoxic properties, which cause it to heal light cases of tuberculosis
and to stay the progress of severe and advanced cases of the disease.
Having now particularly described and ascertained the nature of this invention
as communicated to me and in what manner the same is to be performed, I declare
that what I claim is :-
1. The process of manufacturing from goats or other animals, serum for the cure or
retardation of the progress of tuberculosis, which process consists in injecting
subcutaneously into the living animal first tuberculin, secondly a comparatively large
quantity of tuberculin freed from glycerine and thirdly a sterilized cultivation of
tubercle bacilli containing the dead remains of the bacilli, and in then, after an interval
of several weeks, drawing blood from the animal and allowing this blood to coagulate
whereby antitoxic serum may be separated substantially as set forth.
2. The process of manufacturing from goats or other animals serum for the cure or
retardation of the progress of tuberculosis, which process consists in injecting
subcutaneously into the living animal a large quantity of tuberculin freed from
glycerine and in then injecting a sterilized cultivation of tubercle bacilli containing
the dead remains of the bacilli, and in then after an interval of several weeks drawing
blood from the animal and allowing this blood to coagulate whereby antitoxic serum
may be separated substantially as set forth.
3. As a new product, a serum for the cure or retardation of the progress of
tuberculosis, said serum being obtained by injecting subcutaneously into a living
animal first tuberculin, secondly tubercalin freed from glycerine, and thirdly a sterilize
cultivation of tubercle bacilli containing the deal remains of the bacilli and by
subsequently withdrawing blood from the animal and allowing this blood to coagulate,
substantially as set forth.
4. As a new product, a serum for the cure or retardation of the progress of
tuberculosis, said serum being obtained by injecting subcutaneously into a living
animal first tuberculin freed from glycerine and secondly a sterilized cultivation of
tubercle bacilli containing the dead remains of the bacilli and by subsequently
withdrawing blood from the animal and allowing this blood to coagulate, substantially
as set forth
5. In the manufacture of remedial serum the use of tuberculin freed from glycerine

page_text: for rendering animals immune to tuberculosis substantially as and for the purpose
described.
6. The manufacture of reme lial serum substantially as and in the manner
hereinbefore described.
Dated this 28th day of October 1896
J. C. MEWBURN & ELLIS,
55 and 56 Chancery Lane, London, Chartered Patent Agents.