NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
47
chest, and abdomen rigid, and jaws firmly clenched. It remained in this condi
-tion until the 21st when it died.
Immediately after death anærobic cultivations were made from subcutane
ous tissue taken from the seat of injection of spores, and seat of injection of quinine,.
and also from the liver and the spleen.
A growth of tetanus bacilli was obtained from the seat of injection of spores,
and seat of injection of quinine, but not from the liver or the spleen.
No. 4 Monkey .—(Control of "washed spores " used.)
This monkey was given ½ c.c., "washed tetanus spores" hypodermically in
the right hind leg on September 2nd. The spores were from the same strain and
culture of tetanus as those used for the other three monkeys, and prepared at the
same time, and in the same way. No quinine was given. This animal remained
well.
No. 5 Monkey.—Control of the culture of tetanus from which the " washed
spores" were prepared.
On September 2nd, 1 c.c. of tetanus culture was given hypodermically in the
right hind leg.
Tetanus developed on September 4th, and death took place the next day.
The disease commenced with contraction of the masseter muscles (lockjaw),
difficulty of swallowing, and rapidly advanced to generalised tetanus, and before
death the animal was bent back (opisthotonos).
The rapid course of the disease was no doubt due to the amount of toxine con
- i n e d i n 1 c . c . o f t h e c u l t u r e u s e d . T h e t e t a n i c s y m p t o m s w e r e n o t a n y m o r e
marked in the inoculated hind leg than in the other leg.
Immediately after death anærobic cultivations were made from the subcutane
o u s t i s s u e t a k e n f r o m t h e s e a t o f i n j e c t i o n o f t h e t e t a n u s c u l t u r e , a n d a l s o f r o m
the liver and the spleen.
A growth of tetanus bacilli was obtained from the seat of injection of culture,.
but not from the liver or the spleen.
It will. be seen that the results of the experiments on these three monkeys
which contracted tetanus after inoculation of "washed tetanus spores" in the hind
leg, and hypodermic injections of quinine on front of the chest, harmonise with
similar experiments on guinea pigs. The incubation period was more prolonged
in the case of the monkeys, and when symptoms set in they resembled those found
in the cases of tetanus in the human subject, lockjaw being an early and prominent
symptom, and local tetanus at the site of inoculation being absent (as an early
symptom at any rate) in all three cases, whereas it is always the first symptom in
guinea pigs, and when not lethal, is often the only symptom.
In these cases the infecting micro-organism was recovered from the seat.
of injection of quinine and also from the seat of injection of spores, but not from
H
49
and XII, that there is direct experimental proof to the effect that pure tetanus.
spores, free from toxine and other irritants, fail to give tetanus to guinea pigs and.
monkeys when injected hypodermically ; but in a large percentage of these animals
when quinine is given hypodermically, and in a part of the body far removed from
the spores, tetanus is the result, although the quinine, syringe, and fluid in which
the quinine was dissolved were beyond suspicion, and contained not a trace of
tetanus infection.
The object of this series of experiments was to demonstrate whether tetanus
bacilli can be cultivated from the site of the hypodermic injection of pure " washed
tetanus spores " into guinea pigs, after various periods had elapsed since the spores.
were injected.
Cultivations were made from eight guinea pigs. Most of these animals had
been used as controls in several other experiments in which quinine had been
given in addition to spores. None of them had shown any signs of tetanus and all
were healthy and full grown. The time which elapsed between the date of injec
- t i o n o f p u r e s p o r e s , a n d t h e d a t e o n w h i c h c u l t i v a t i o n s w e r e m a d e f r o m t h e s i t e
into which the spores had been injected, varied from five weeks to seven months ; so
that ample time had elapsed to allow of the spores producing infection, or being dis
- p o s e d o f b y p h a g o c y t o s i s , o r s t o w e d a w a y l o c a l l y i n t h e t i s s u e s a t t h e s e a t o f
injection, and alive and capable of producing tetanus should conditions become
favourable.
The animals were killed with chloroform. The skin at the site of injection of
spores (right hind leg in all cases) was rendered aseptic, cut into, and a small
portion of the subcutaneous tissue and adjacent muscles removed, and placed in
broth tubes. The tubes were then rendered anærobic, and incubated at a tem
perature of 37°C., for a week at least. In all these eight experiments true tetanus
bacilli ware isolated, and proved virulent by giving tetanus to other guinea pigs.
It is evident from these results that tetanus spores can remain localised in the
tissues for months. We know from experiments carried out years ago by Metch
-nikoff, Vincent and others, that when washed tetanus spores are injected hypo
- d e r m i c a l l y i n t o a n i m a l s t h e p h a g o c y t e s p i c k t h e m u p a n d k i l l t h e m o f f , a n d
that the spores can be demonstrated inside phagocytes taken from the site soon
after injection. Some of the spores must escape being efficiently disposed of by
the phagocytes, otherwise it would be impossible to obtain evidence of their pre
- s e n c e i n t h e t i s s u e s m o n t h s a f t e r w a r d s . N o d o u b t m o s t o f t h e m a r e d e s t r o y e d b y
the phagocytes, and those which escape get fixed in a safe place in the tissues,
and remain there without giving rise to any symptoms.
When a person contracts tetanus through a wound, it means that tetanus
spores have entered the wound, and found suitable conditions in which to ger
- m i n a t e a n d g i v e r i s e t o t h e d i s e a s e .
H 2
50
On the other hand, should the local conditions be unfavourable for the tetanus
spores to germinate, and should they escape phagocytosis, what is to prevent them
remaining stowed away and locked up in the tissue locally for months after the wound
has healed ? A condition of this kind would correspond to what is found in guinea
pigs, when washed tetanus spores are injected locally, except that in a tetanus-
infected wound, the chances are more in favour of the spores finding suitable condi-
tions for germination on account of other wound-infecting organisms accom-
panying them. There is still another condition necessary when a wound is infected
with tetanus spores, viz., anærobic conditions. I have no doubt that in many
tetanus-infected wounds, owing to the absence of anærobic conditions, tetanus fails
to set in, and that the spores which escaped phagocytosis get hidden away in the
tissues locally.
Table XIII.
Experiments to test whether Tetanus Spores can remain alive and localised at the site of
a subcutaneous injection in Guinea Pigs.
No of
experi-
ment.
Date and site of injection of
spores.
Date on which the
animal was chloro-
formed and tissue
taken from site of
injection for making
cultivations.
Result of anærobic cultiva-
tions in broth.
REMARKS.
1
30th August 1910, hind leg . 30th September 1910 Growth of tetanus bacilli . The tetanus bacilli
isolated from this
series of experi-
ments were viru-
lent for guinea
pigs.
2
27th August 1910, hind leg .
D i t t o
.
Ditto.
3
Ditto ditto.
D i t t o
.
Ditto.
4
Ditto ditto.
D i t t o
.
Ditto.
5
7th April 1910, hind leg
. 3rd October 1910 .
Ditto.
6
9th April 1910, hind leg
.
D i t t o
.
Ditto.
7
30th August 1910, hind leg .
D i t t o
.
Ditto.
8
Ditto ditto.
D i t t o
.. Ditto.
The results of this series of experiments prove the possibility of tetanus spores remaining latent and localised
in the tissues for months.
THE 14TH SERIES OF EXPERIMENTS.
In the experiments given, advantage was taken, on the death of a monkey
and two guinea pigs from acute tetanus, to prepare anærobic cultivations from
blood taken from the heart, and at the same time to inoculate three other guinea
pigs with blood also taken from the heart of these three cases of acute tetanus.
The blood was taken immediately after death and before it had had time to
clot.
No. 1 received 1 c.c. from a tetanus guinea pig.
51
No. 2 received 2 c.c., from another tetanus guinea pig.
No. 3 received 3 c.c., from a tetanus monkey.
The anærobic blood cultivations gave negative results in all three cases ; no
tetanus bacilli appeared in any of them.
Nos. 1 and 2 guinea pigs remained well.
No. 3 guinea pig inoculated with 3 c.c., of monkey's blood remained well for
a week, when rapid wasting set in, and it died on the 12th day. It showed no symp-
toms of tetanus at any time. An anærobic cultivation made from the site of in-
jection of blood failed to grow tetanus bacilli.
In these three cases of acute tetanus the blood furnished no evidence of
containing tetanus infection.
Table XIV.
Experiments to prove whether the blood in case of acute Tetanus contains infection at
the time of death.
No. of
experi-
meat.
Source from which the
blood was taken.
Result of animal experi-
meat with blood.
Result of anærobic
cultivation made
from blood.
REMARKS.
1
A guinea pig which had
died from severe and
well marked experi-
mental tetanus ; blood
was taken from the
heart immediately
after death.
1 c.c. injected subcuta-
neously in the hind
leg of a guinea pig
failed to give the
animal tetanus.
½ c.c. transferred to
a broth tube, and
incubated anærobic-
ally at a tempera-
ture of 37°C for 10
days gave negative
results.
The two guinea pigs and the
monkey from which blood
was taken for these experi-
ments were typical severe
cases of experimental tetanus,
and in each of them a cultiva-
tion made after death from
the site of inoculation gave a
growth of tetanus bacilli. It
is evident from these results
that the blood in a case of
tetanus does not contain any
living tetanus spores, or
tetanus bacilli at the time of
death.
2
Ditto
. 2 c.c. injected subcuta-
neously in the hind
leg of a guinea pig
failed to give the
animal tetanus.
Ditto.
3
A monkey which had
died from severe and
well marked experi-
mental tetanus ; blood
was taken from the
heart immediately
after death.
3 c.c. injected subcuta-
neously in the hind
leg of a guinea pig
failed to give the
animal tetanus.
Ditto.
The results of this series of experiments show that the blood in cases of acute tetanus does not contain infection
at the time of death.
THE 15TH SERIES OF EXPERIMENTS.
Considering the fact that tetanus is the most widely distributed of all patho-
genic micro-organisms, and that it has been isolated from the intestinal canal of
horses, sheep, cattle, and other animals, it is only reasonable that one would ex-
pect to find a similar infection of the intestinal tract of men. The fact that it is
present in the intestinal tract of man and has been isolated from human excreta
52
is mentioned in most text books on bacteriology, but I cannot find any information
about the percentage of cases in which it is present, or whether tetanus bacilli
from this source have ever been proved virulent, and capable of giving tetanus to
susceptible animals. With a view to gaining some information on these two points
the series of experiments detailed in Table XV were carried out.
Ten samples of fæces were obtained haphazard from 10 separate sources and
from healthy individuals.
In each case an emulsion was made by adding about 2 grammes to 15 c.c. of
sterile normal saline solution. After thoroughly mixing, ½ c.c. was filled into a glass
pipette and heated in a water bath to a temperature of 80° C. for 5 minutes. After
heating, the contents of the capsule were transferred to an ordinary broth tube,
which was made anærobic, and incubated at a temperature of 37°C. for a week..
At the end of this time tetanus bacilli when present were easily distinguished by
staining specimens.
In no case was this sufficient to obtain a pure growth, as many other spore
bearing anærobic bacilli found in the intestinal contents of man survive heating
for 5 minutes at a temperature of 80°C., and as a matter of fact, in every instance
there was evidence that such bacilli were present when the tubes were examined.
In all ten cases pseudo-tetanus bacilli were found, and in four out of the ten cases
true tetanus bacilli were also found.
The pseudo-tetanus bacillus found in these cases might, on a casual glance
and without experience, be mistaken for true tetanus bacilli ; but on a close ex
-amination there is a marked difference. In the true tetanus bacillus when spores
have formed they are round, and at the very end of a thin straight rod. In the
psuedo-tetanus bacillus when spores have formed they are slightly oval, not
quite at the end of the bacillus, which is shorter and stouter than the true tetanus
bacillus, and the end opposite to the spore is often pointed. When the culture is
a few days old, the rod part of the pseudo-tetanus bacillus disappears, and spores
only are found ; whereas in the true tetanus bacillus, the spores remain on the ends
of the rods for weeks. Finally the pseudo-tetanus bacillus also grows anærobically,
and is non-pathogenic for animals.
Bacilli answering in every detail to the description of true tetanus bacilli were
isolated in four out of the ten cases in which an attempt was made.
The virulence of these four cultures was tested on guinea pigs, and with the
following results : Two of the cultures were virulent ; 1 c.c. of a 14 days' growth
produced marked tetanus in guinea pigs in 20 hours and death in 42 hours. One
culture was only slightly virulent as it required 48 hours for 1 c.c. of a 14 days'
culture to produce local tetanus in a guinea pig. The fourth culture was non
-virulent-1 c.c. of a 14 days' growth failed to give local tetanus to a guinea pig.
The very fact that tetanus spores are to be found in the intestinal tracts of a
53
large percentage of individuals opens up the possibility of so-called idiopathic teta-
nus occurring, provided the spores are virulent and get lodged in a focus of dead tis-
sue, and under anærobic condition. Granted that it is possible for tetanus spores to
enter the circulation from the intestinal tract where the mucous membrance is in-
jured, ulcerated, or in any way damaged to such an extent as to be incapable of keep-
ing back infection, what is to prevent such spores getting stranded in an injury
somewhere else in the body where there is dead anærobic tissue? The hypodermic
or intramuscular injection of quinine would prepare a suitable focus in a case of this
kind, and so would injuries of various sorts which are constantly occurring. It seems
to me that this is a reasonable possibility of infection in cases of so-called idiopathic
tetanus, and in many other injuries including quinine injections, but to say more
on the subject at present would savour of conclusions arrived at in the absence of
proof.
Table XV.
Experiments proving the presence of Tetanus germs in normal human fæces.
No. of
experi-
ment.
Preparation of mate-
rial used.
Cultivation used.
Result.
REMARKS.
1
Emulsion of normal
fæces in normal
saline solution and
heated 5 minutes
at 80°C.
Anærobic broth
culture.
Growth of tetanus bacilli In the four specimens which con-
tained true tetanus bacilli, there
were also other spore-forming
bacilli, including the pseudo-
tetanus bacilli described by Tavel
and Zimmerman, but these
were easily distinguished
from the true form. Four
out of the six negative spe-
cimens contained pseudo- tetanus
bacilli, and other spore-forming
germs ; numerous spore-forming
bacilli with no resemblance to
either the true or pseudo tetanus
bacillus were found in the re-
maining two specimens.
2
Ditto
.
Ditto
.
Ditto.
3
Ditto
.
Ditto
. Negative.
4
Ditto
.
Ditto
.
Ditto.
5
Ditto
.
Ditto
.
Ditto.
6
Ditto
.
Ditto
. Growth of tetanus bacilli.
7
Ditto
.
Ditto
. Negative.
In experiments Nos. 1, 6 and 9 the
tetanus bacilli isolated were viru-
lent for guinea pigs in small doses.
8
Ditto
.
Ditto
.
Ditto.
9
Ditto
.
Ditto
. Growth of tetanus bacilli.
In experiment No. 2 the tetanus
bacilli isolated were non-virulent
for guinea pigs in a dose of 1 c.c.
of a 14 days' culture.
10
Ditto
.
Ditto
. Negative.
The experiments in this table prove that a large percentage of normal healthy persons harbour virluent tetanus
bacilli in their intestinal tracts (4 out of 10, or 40 per cent.).
THE 16TH SERIES OF EXPERIMENTS.
This series of experiments was primarily intended to serve as a control of the
tetanus bacilli isolated from the site of quinine injections in guinea pigs and monkeys
in the experiments recorded in Tables VIII, IX and XII in which quinine was given
hypodermically on front of the chest, and "washed tetanus spores" or tetanus
cultures in the hind leg.
55
Table XVI.
Experiments on Guinea Pigs to determine whether Tetanus bacilli can be cultivated from
the site of hypodermic injections of Quinine in cases where no Tetanus spores or
Tetanus cultures had been given.
No . f
o
experi-
ment.
Dates on which
quinine was
given hypoder-
mically.
Site of injection
of quinine.
Date on which
anærobic culti-
vations were
made from site
of quinine
injection.
Result.
REMARKS.
1
6th October 1910,
¾ gr.
8th October 1910,
¾ g r .
Front of chest . 10th October
1910.
Negative
.
The cultivations from Nos.
1, 2 and 3 experiments
were examined on several
occasions up to the 14th
day, and in none of them
were there any bacteria
resembling true tetanus
bacilli.
2
Di t t o
.
Ditto
.
Ditto
.
Ditto.
3
Ditto
.
Ditto
.
Ditto
.
Ditto
4
Di t t o
.
Ditto
.
Ditto
. Growth of teta-
nus bacilli.
In the cultivation from No. 4
experiment, a pure growth
of tetanus bacilli was obtain-
ed on the third day. This
culture was very virulent, as
evidenced by the fact that
0.1 c.c. of a 6 days' growth
was sufficient to produce
tetanus in a guinea pig in 12
hours and death in 24 hours.
THE 17TH SERIES OF EXPERIMENTS.
In the experiments on guinea pigs and monkeys recorded in Tables VIII, IX
and XII, tetanus bacilli were isolated from the site of hypodermic injections of quinine
given on front of the chest, when tetanus cultures, or "washed tetanus spores"
had been injected in the hind leg.
The only reasonable conclusion to be arrived at in order to explain the presence
of tetanus bacilli at the site of quinine injections in the experiments referred to is,
that they had been transferred from the site of infection in the leg to the site of
quinine injections on the chest ; and with the object of controlling this conclusion
the following experiments were carried out.
Four healthy full grown guinea pigs were each given a hypodermic injection of
¾ grain quinine on front of the chest, and after an interval of two days the dose was
repeated. Two days after the second dose the animals were killed with chloroform
and anærobic cultivations made from the subcutaneous tissue at the site of quinine
injections on front of the chest, and also from the intestinal contents.
No evidence was obtained of the presence of tetanus infection in any of these
cultivations.
The results of this series of experiments prove that the conclusion arrived at
to explain the presence of tetanus bacilli isolated from the site of quinine injection
in the experiments recorded in Tables VIII, IX and XII is probably correct.
I
56
Table XVII.
Experiments on Guinea Pigs to determine whether Tetanus bacilli can be cultivated from
the site of hypodermic injections of Quinine, or from the intestinal tract in cases
where no Tetanus spores, and no Tetanus cultures had been given.
No. of
experi-
ment.
Dates on
which quinine
was given
hypodermical-
ly.
Site of
injection
of
quinine.
DATES ON WHICH ANÆROBIC
CULTIVATIONS WERE MADE
FROM
RESULT OF ANÆROBIC CULTI-
VATIONS MADE FROM
REMARKS.
Site of quinine
injection.
Intestinal
tract.
Site of quinine
injection.
Intestinal
tract.
1 15th and
17th October
1910, ¾ gr.
each time.
Front of
chest.
19th October
1910.
19th October
1910.
Tetanus bacil-
li negative.
Tetanus bacil-
li negative.
2
Ditto
. Ditto . Ditto
. Ditto
.
Ditto .
Ditto.
3
Ditto
. Ditto . Ditto
. Ditto
.
Ditto .
Ditto.
4
Ditto
. Ditto . Ditto
. Ditto
.
Ditto .
Ditto.
THE 18TH SERIES OF EXPERIMENTS.
The result of No. 4 experiment in Table XVI rendered it necessary to prove
whether guinea pigs harbour tetanus infection in their intestinal tract. Ten healthy
guinea pigs were killed with chloroform, and a portion of the contents of the rectum
and large intestine removed and emulsified in sterile broth, 1 c.c. from each emul-
sion was filled into glass pipettes, and heated in a water bath for 5 minutes at a tem-
perature of 80ºC., and used to inseminate 10 broth tubes. When these tubes
had been incubated for 10 days at a temperature of 37ºC., and under anærobic
conditions, tetanus bacilli were found in one case, but not in the other nine. In
three of the ten cases examined pseudo-tetanus bacilli were found ; and other
spore-forming bacilli were present in all. The tetanus bacilli found in the one case
were probably non-virulent, as 1 c.c. of a 10 days' culture failed to give a guinea
pig tetanus.
It is evident from the result of this series of experiments that only about 10
per cent. of guinea pigs harbour tetanus spores in their intestinal tract ; and that
these spores are not in all cases virulent. This possibly accounts for the fact that
in numerous experiments on these animals with hypodermic injections of quinine
only, none of them contract tetanus.
On the other hand the result of No. 4 experiment in Table XVI would point
to the possibility of virulent spores being present in the intestinal tract of
guinea pigs in exceptional cases.
57
Table XVIII.
Experiments to determine whether normal healthy Guinea Pigs harbour Tetanus spores
in their intestinal tract.
No. of
experi-
ment.
Preparation of material
used.
Cultivation used.
Result.
REMARKS.
1
Emulsion in normal sal-
ine solution from por-
tions of the rectum and
the cæcum. Emulsion
was heated for 5 minutes
at a temperature of
80° C.
Anærobic broth
culture
Negative.
2
Ditto
.
Ditto
. Growth of tetanus
bacilli.
Non-virulent for guinea
pigs in a dose of 1 c.c. of a.
10 days' culture.
3
Ditto
. .
Ditto
. Negative.
4
Ditto
.
.
Ditto
.
Ditto.
5
Ditto
.
.
Ditto
.
Ditto.
6
Ditto
.
.
Ditto
.
Ditto.
7
Ditto
.
.
Ditto
.
Ditto.
8
Ditto
.
.
Ditto
.
Ditto.
9
Ditto
.
.
Ditto
.
Ditto.
10
Ditto
.
.
Ditto
.
Ditto.
THE 19TH SERIES OF EXPERIMENTS.
The object of this series of experiments was to test whether antitetanic serum
can be trusted to protect against a certain lethal dose of a virulent tetanus culture,
followed next day by a hypodermic injection of quinine. The tetanus culture used
as a test was extremely virulent : 1/50 c. c. was sufficient to kill a full grown guinea
pig in 24 hours. It contained well formed bacilli with spores and toxine.
When a culture of this kind is injected hypodermically into a guinea pig the
toxine is soon absorbed, and the spores proceed to germinate into fresh bacilli,
which in their turn elaborate more toxine, and so the disease advances unchecked ;
but when antitetanic serum has been given before the tetanus culture, the toxine
is neutralised, and a passive immunity is conferred which prevents the spores from
developing, and leaves them an easy prey to the phagocytes. Two guinea pigs were
each given 3 c.c. of tetanus antitoxin hypodermically, on the front of the chest,
and after 3 hours a dose of a virulent tetanus culture in the hind leg. Next day
they were given ¾ grain quinine hypodermically in the side. Both remained well.
A control guinea pig which received half the amount of the same tetanus culture
I 2