NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
27
1.
Quinine.—When quinine is injected hypodermically into guinea pigs, it soon
becomes absorbed, but immediately on injection and before absorption has taken
place, the tissues at the site of injection are severely damaged. This local destruc-
tion of tissues is so complete that in most cases a slough forms, which involves the
skin and comes to the surface, leaving an open sore within a few days from the time
the quinine was given. The acidity of the solution may to a certain extent account
for this local action, but it happens also when "Quinoforme Lacroix," a patent
French preparation absolutely neutral in reaction, is given.
The effect of a localised necrotic subcutaneous focus would be to establish a
suitable anærobic medium for the growth of tetanus spores should they by any
chance become lodged there. At the same time, one of the immediate effects of a
large dose of quinine would be to produce a more or less paralysing effect on the
phagocytes generally, and a negative chemiotaxis on the phagocytes locally,
conditions which would still further the growth of any tetanus spores which
happened to get lodged at the spot.
2. "Washed tetanus spores."—Vaillard and Vincent 15 have proved that when
"washed tetanus spores" are injected hypodermically into guinea pigs, they are
picked up and digested by the phagocytes which crowd to the seat of injection,
provided there is nothing introduced with the spores to destroy tissue, produce a
negative chemiotaxis, or in any way to divert the phagocytes from efficiently
performing their function of bactericidal agents.
It may happen even with active phagocytes that some of the spores escape
destruction, and become stowed away locally, to grow and produce infection at some
future time when conditions become favourable. It may also happen that some of
the phagocytes require a considerable time to kill off and digest the toughest of the
spores, and that phagocytes in this condition on re-entering the circulation may get
stranded or lodged in a suitable site where the spores can grow and give rise to
tetanus. The subcutaneous or intramuscular injections of quinine would produce
such foci provided spore-laden phagocytes become lodged there.
On the assumption that this is what happens when quinine and tetanus spores
are injected into separate parts of the body, one would expect more infections in
susceptible animals when the quinine is injected on the day before, or at the same
time as the spores, and fewer infections when the spores are injected a day or more
before the quinine. Also one would expect never to fail in producing tetanus
when solutions of quinine or other irritants such as lactic acid are injected mixed
with the spores.
On the other hand, one would expect to fail in producing tetanus when non-
irritants (which become rapidly absorbed), such as morphia or sterile normal saline
solution, are injected mixed with washed tetanus spores. As a matter of fact the
accuracy of these conjectures is verified by experimental tests as will be seen by
28
referring to the numerous experiments detailed in the succeeding part of this paper.
In order to bring facts to bear upon what has been briefly outlined in the fore-
going remarks, it will be necessary to take up and deal ser iatim with the experi-
ments upon which these conjectures and explanations are based.
In the first and second series of experiments the virulence of the strain of
tetanus used had become attenuated, and the subsequent experiments were
carried out with another strain, which possessed a higher degree of virulence, but
even this strain was not very virulent.
Care was taken to use only absolutely pure cultures. The purity of every
culture used was controlled by examining stained specimens under the microscope,
and planting out fresh cultures. In every case in which "washed spores" were used,
a control cultivation was made to prove that the spores were alive and pure. The
" w a s h e d s p o r e s " w e r e u s e d i m m e d i a t e l y a f t e r t h e i r p r e p a r a t i o n , i n o r d e r t o a v o i d
any subsequent chance of growth or contamination taking place, as either of these
mishaps would have vitiated the experiments. This necessitated the preparation
of a large number of tetanus cultivations, so as always to have a culture at a
suitable stage of growth when required.
The "washed spores" were prepared as follows : Ten c.c. anærobic broth
cultures which had been grown for a month at a temperature of 37° C. were used
; 2 c . c . w e r e p i p e t t e d o f f f r o m t h e b o t t o m o f a c u l t u r e a n d t r a n s f e r r e d t o a s t e r i l e t e s t
tube, and then heated in a water bath at a temperature of 75° C. for 5 minutes.
Before heating, a specimen was stained and examined to test for purity, and also a
cultivation made so as to be available when required. Another cultivation was
made after heating to prove that the spores were alive, and of course this culture
could also be utilised when it had sufficiently grown. When broth cultures of
tetanus have been allowed to incubate at a temperature of 37° C. for a month, the
spores are well formed and resistant, and by that time they have sunk down to the
bottom of the tube, so that, provided the tube has not been shaken, it is possible
practically to remove nearly all the spores by pipetting off the fluid from the
bottom part of the cultivation.
When 2 c.c. are pipetted off from a 10 c.c. culture for heating purposes,
and when the same quantity of this heated fluid containing spores is used for a series
of experiments, each animal experimented on receives practically the same amount
o f s p o r e s ; t h i s i s w h a t w a s d o n e i n a l l c a s e s i n w h i c h w a s h e d s p o r e s w e r e u s e d .
Guinea pigs received 0.25 c. c. and in the case of monkeys ½ c. c. was given.
The strictest aseptic precautions were observed in the preparation and admin-
istration of quinine, spores, tetanus cultures, or other materials used, and in no
instance were control experiments omitted.
The animals experimented upon were in most cases full grown, and in all
cases healthy, they were kept isolated in separate cages, and under the observation
29
of trustworthy attendants night and day. At night they were visited every two
hours by a man on duty, and during the daytime they were under my own observa-
tion, but there was always an attendant on duty to note down anything that hap-
pened and bring it to my notice.
The details of these experiments are plainly given in the Tables in which they
are recorded, so that only a few remarks are necessary to draw attention to each
series separately.
30
CHAPTER V.
A Series of Experiments recorded in Tables I to XIX.
THE 1ST SERIES OF EXPERIMENTS.
In this series four guinea pigs were inoculated with " washed tetanus spores "
under the skin of the hind leg, and next half a grain of quinine was injected hypoder-
mically on the front of the chest, and repeated for three days. On the fourth day
one animal developed tetanus, and died three days later. The other three animals
remained well. Two control guinea pigs inoculated with " washed tetanus spores "
only, remained well. Another control inoculated with 0.5 c.c. of the tetanus
culture from which the spores were prepared developed tetanus on the fourth day,
and died next day.
It will be seen from this last experiment that the tetauns culture used was not
very virulent, otherwise it would not have required four days for 0.5 c.c. to
produce tetanus in a guinea pig.
The results of the experiments recorded in this table show that when quinine
is injected hypodermically into guinea pigs it produces conditions favourable for
infection with " washed tetanus spores, " even when the spores are of low virulence.
Table I.
Experiments on Guinea Pigs to demonstrate the effects of Quinine in producing favour-
able conditions for infection with " Washed Tetanus Spores, " when Spores are
g i v e n o n e d a y b e f o r e Q u i n i n e .
No. of ex-
periment.
Date of injec-
tion of spores.
Date of injec-
tion of quinine.
Amount o f
quinine
injected.
Result.
REMARKS.
gr.
1
.
. 1st March
1910, right
hind leg.
2nd March
1910.
1
Tetanus, 5th March. Symptoms local at outset,
then became general. An
anærobic culture made
from the tissues at the
site of injection of spores
grew tetanus bacilli.
Died, 8th March.
3rd March
1910.
½ on front of
chest.
4th March
1910.
½
2
.
. Ditto .
. Ditto
. Ditto . Remained well.
3
.
. Ditto .
. Ditto
. Ditt
.
Ditto.
4
.
. Ditto .
. Ditto
. Ditt
.
Ditto.
5 and 6 .
(Control of
spores.)
Ditto .
.
..
..
Both controls re-
mained well.
7 (Control of
the culture
from which
spores were
prepared.)
0.5 c.c. tetanus
culture, 1st
March.
Tetanus, 4th March. Symptoms local at first,
then became general.
Died, 5th March.
The culture of tetanus used for this series of experiments was of low virulence, as evidenced by the fact
that three days elapsed before ½ c.c. produced tetanus in a guinea pig. (See No. 7 control experiment.) This series
shows 25 per cent. of infections when spores were given one day before quinine; the spores were injected in the
hind leg, and quinine on front of the chest.
31
THE 2ND SERIES OF EXPERIMENTS.
A series of experiments in which quinine injections were commenced one day
before spores were given.
Three guinea pigs were each given half a grain of quinine hypodermically on
the front of the chest, and next day " washed tetanus spores " hypodermically in
the hind leg. The quinine injections in ½ grain doses were continued for a
week.
On the 10th day from the time the spores were given one animal developed
tetanus in the leg into which the spores had been injected, and next day the tetanus
symptoms had extended to the side, and the disease became chronic, and lasted
for a month, when recovery took place.
The other two animals remained well, as also did the control guinea pig which
received spores only.
The control which received 0.5 c. c. of the culture from which the spores were
prepared contracted tetanus on the second day, and died three days later.
The results of the experiments recorded in this table show that when quinine
is injected hypodermically into guinea pigs it produces conditions favourable for
infection with " washed tetanus spores."
Table II.
Experiments on Guinea Pigs to demonstrate the effects of Quinine in producing favour-
able conditions for infection with " Washed Tetanus Spores " when Quinine is
commenced one day before Spores are given.
No. of ex-
periment.
Date of injec-
tion of spores.
Date of injec-
tion of quinine:
Amount of qu-
nine injected.
Result.
REMARKS.
1
.
. 4th March
1910, right
hind leg.
3rd to 10th
March.
½ gr. daily on
front of chest.
Tetanus, 10th March.
Severe and localised
to right leg and
right side; became
chronic, and after
a month recovered.
Disease localised to the
side on which spores were
injected.
2
.
.
Ditto
.
Ditto
.
Ditto
. Remained well.
3
.
.
Ditto
.
Ditto
.
Ditto
.
Ditto.
4 (Control of
spores).
Ditto
.
..
. .
Ditto.
5 (Control of
the cul-
ture from
which sp-
ores were
prepared.)
0.5 c.c. teta-
nus culture,
4th March.
..
..
Tetanus, 5th March. Symptoms local at first,
then became general.
Died, 8th March.
This series shows 33.3 per cent. of infections when quinine was commenced one day before spores were given:
the spores were injected in the hind leg, and quinine on front of the chest.
32
THE 3RD SERIES OF EXPERIMENTS.
This series is a repetition of the 2nd series, but a more virulent strain of tetanus
culture was used for the preparation of the " washed spores."
Four guinea pigs were given 1/2gr. quinine hypodermically on front of the chest,
and next day " washed tetanus spores " hypodermically in the right hind leg. The
quinine treatment was continued daily in ½g r . d o s e s i n o n e a n i m a l f o r t h r e e d a y s ,
and in the other three for five days. No. 1 which had quinine for three days developed
tetanus on the third day after spores were given and died on the fifth day. No. 2
developed tetanus on the fifth day after spores were given, and died on the thirteenth
day. Nos. 3 and 4 developed tetanus on the sixth day after spores were given, and
both these animals passed through a severe attack of local tetanus, which involved
the right leg and right side of the body for three weeks, when they eventually
recovered.
Two control guinea pigs inoculated with " washed spores " only, remained well,
and the control culture guinea pig developed tetanus after 24 hours and died next
day .
In the No. 2 guinea pig of this series, local tetanus developed on the fifth day
after spores were given, and the disease remained localised to the leg inoculated
with spores for nine days, after which acute generalised tetanus set in, and the animal
died two days later. Immediately after death, anæerobic cultivations in broth tubes
were made from seat of injection of spores (right hind leg) ; seat of injection of
quinine (chest) ; spleen ; liver ; lungs ; kidneys ; heart's blood. When these cul-
tivations had been incubated for a month at 37°C. they were opened and examined
with the following results :
—
A rich growth of tetanus bacilli with well formed spores from the seat
of injection of quinine on the chest, and also from the seat of injection
of spores in the leg. A few micrococci in the cultivation from the
lungs, and the remaining four cultivations from the liver, spleen,
kidneys, and heart's blood were sterile.
After this result it was deemed advisable to test further whether tetanus germs
can be conveyed from a remote site of infection such as the leg, to another part of
the body such as the chest, where quinine has been injected. The results of the
experiments detailed in Tables VIII, IX and XII prove that this is what happens
in probably every case, and that a hypodermic injection of quinine establishes a very
favourable local site for the growth of tetanus germs should any ever reach the
spot. A phagocyte carrying a few tetanus spores which happened to get stranded
in such a site would prove the starting point of a fresh focus of infection.
Is this what happened in No. 2 guinea pig of this series when it had chronic
and localised tetanus for nine days, and then acute and fatal symptoms suddenly
set in ? In 1904, Vincent, 16 when experimenting with quinine and tetanus infections,
33
also obtained results which favour the view that quinine injections produce foci
favourable for the growth of tetanus bacili, and that infection may be conveyed from
the original site of the disease to these foci.
Table III.
Experiments on Guinea Pigs to demonstrate the effects of Quinine in producing favour-
able conditions for infection with "Washed Tetanus Spores" when Quinine is given
one day before Spores.
No. of
experiment.
Date of
injection of
spores.
Date of
injection of
quinine.
Amount of
quinine
injected.
Result.
REMARKS.
1.
7th April 1910,
right hind leg.
6th to 9th
April.
½ gr. daily on
front chest.
Tetanus, 10th April.
Died, 12th April.
Symptoms local at outset,
then became general.
*2
Ditto
.
Ditto
.
Ditto
. Tetanus, 12th April.
Died, 23rd April.
Symptoms local at first,
advanced slowly, then
became severe and gene-
ral the day before death.
See footnote*.
3.
Ditto
.
Ditto
.
Ditto
. Tetanus, 13th April.
Severe, but even-
tually recovered
after three weeks.
Symptoms localized to
right leg and right side
of body.
4.
Ditto
.
Ditto
.
Ditto
. Same as experiment
No. 3.
Ditto ditto.
5 and 6 (Con-
trols of
spores.)
Ditto
.
...
. . .
Both controls re-
mained well.
7 (Control of
cu1ture
used for
prepara-
tion of
spores.)
0.25 c.c. o
f
culture on
7th April.
. . .
. . .
Tetanus, 8th April.
Severe. Died, 9th
April.
Symptoms local at outset,
but soon became general.
This Table shows 100 per cent. of infections when quinine is commenced one day before spores are given.
* Experiment No. 2.
As the disease in this guinea pig assumed a somewhat chronic and localised character for nine days at the outset,
and then became acute and general, anærobic cultivations in broth were made from the following tissues and locali-
ties immediately after death, to prove whether infection had been transferred further than the seat of injection of
spores.—Seat of injection of quinine; seat of injection of spores; spleen; liver; lungs; kidney; heart's blood.
Result. —After incubating at 37°C. for a month, there was a rich growth of tetanus bacilli from the seat of
injection of spores and also from the seat of injection of quinine. All other tubes remained sterile, with the excep-
tion of the one from the lungs which grew micrococci only.
The skin at the site of the quinine injections was unbroken at the time of death, but there was subcutaneous
evidence of the escharotic action of the quinine.
In this case infection was conveyed from the seat of injection of spores in the hind leg, to the seat of injection
of quinine on front of the chest.
THE 4TH SERIES OF EXPERIMENTS.
A repetition of the experiments recorded in Tables II and III. Quinine was
given hypodermically on the front of the chest to four guinea pigs one day before.
F 2
34
"washed tetanus spores " were given hypodermically in the hind leg, and two out of
the four animals treated in this manner developed tetanus; one on the fifth day
after the spores were given, the other on the sixth day.
The one which developed tetanus on the sixth day died three days later. Anæ-
robic cultivations made from the site of spores, and the site of quinine grew tetanus
bacilli; but those from the liver and spleen gave negative results.
The other guinea pig remained in a state of chronic tetanus for five weeks, and
eventually recovered. The control guinea pig which received " washed spores"
only, remained well, and the control culture animal developed tetanus on the second
day, and died on the fourth day.
The result of this series of experiments confirms those recorded in Tables II
and III, but the percentage of infections are 50 per cent. as compared with 100 per
cent. in Table III and 33.3 per cent. in Table II.
Table IV.
Experiments on Guinea Pigs to prove the influence of Quinine in producing favourable
conditions for Tetanus infection when Quinine is commenced one day before " Washed
Tetanus Spores " are given.
No. of
experiment.
Date of
injection of
spores.
Date of
injection of
quinine.
Amount of
quinine
injected.
Result.
REMARKS.
1
27th August
1910, right
hind leg.
26th to 28th
August.
½ g r . d a i l y
for two
days on
front of
chest.
Tetanus, 3rd Septem-
ber. Died, 6th
September.
Disease local at first, then
became general. Tetanus
bacilli grew in anærobic
cultivations made from site
of injection of spores, and
from site of injection of
quinine, but not from the
liver, or spleen.
2
Ditto
. Ditto
.
Ditto . Tetanus, 2nd Septem-
ber ; remained chro-
nic for five weeks,
then recovered.
Disease localised to the
right leg, and right side of
body; very severe for a
week, when daily improve-
ment set in.
3
Ditto
. Ditto
.
Ditto . Remained well.
4
Ditto
. Ditto
.
Ditto .
Ditto.
5 (Control of
spores.)
Ditto
.
Nil.
Nil.
Ditto.
6 (Control of
culture
from which
spores
were pre-
pared.)
½ c.c. culture,
right hind
leg.
Nil.
Nil.
Tetanus, 28th August.
Died, 30th August.
Local at outset, then became
general.
This Table shows 50 per cent. of infections when quinine is given one day before spores; the quinine was in
jected on front of the chest, and spores in the hind leg.
35
THE 5TH SERIES OF EXPERIMENTS.
The experiments in this series illustrate the effects of quinine in producing
favourable conditions for infection with "washed tetanus" (which are by
themselves non-infective) when injected into guinea pigs under varying con-
ditions.
Six guinea pigs of approximately the same age and size were selected, and
divided into three lots of two each, so as to contrast the effects of quinine when
given:—
1. One day before "washed tetanus spores" were given.
2. Same day and time as "washed tetanus spores" were given.
3. One day after "washed tetanus spores" were given.
The spores were injected hypodermically into the left hind leg and only one
dose given, and the quinine hypodermically on the front of the chest in ½ grain
doses for four days.
In the first lot tetanus set in on the third day from the time the spores were
given, and in the other two lots on the fourth day.
In all these six animals the disease ran a chronic course, and was confined to
the left hind leg (seat of injection of spores), and left side of the body: and for over
a fortnight they were bent to the infected side, and the infected leg stuck out stiff
when they crawled along on being taken out of their cages. After a month they
all eventually recovered. In the two animals which got quinine one day before
spores, the disease was more severe than in the others, and as I have already men-
tioned it set in one day earlier; so that in these two experiments the quinine had
some influence in preparing more favourable conditions for spores to develop, than
in the other two lots in which it was given in one lot same day as spores, and in the
other the day after.
Two control guinea pigs were given spores only, and both remained well. An-
other control guinea pig was given ¼ c.c. of the tetanus culture from which the
spores were prepared, and tetanus set in on the second day, and it died next
day.
The results of this series of experiments in addition to proving the favouring
influence of quinine in producing tetanus when "washed spores" are given, also
prove that an earlier infection is likely to take place when the quinine is given
some time before the spores.
36
Table V.
Experiments to contrast the effects of Quinine on Guinea Pigs when given
(a) One day before "Washed Tetanus Spores" were given.
(b) Same day as "Washed Tetanus Spores" were given.
(c) One day after "Washed Tetanus Spores" were given.
No. of
experiment.
Date and seat
of injection
of spores.
Date and seat
of injection
of quinine.
Amount of
quinine
injected.
Result.
REMARKS.
1
2nd July 1910,
left hind leg.
1st to 4th
July on front
of chest.
½ gr. daily
for 4 days.
Tetanus, 5th July;
severe and confined
to left side. Re-
covered after a
month.
In both these experiments.
quinine was commenced
one day before spores,
and tetanus set in on the
third day.
2
Ditto
.
Ditto
.
Ditto
. Same as experiment
No. 1.
3
Ditto
. 2nd to 6th
July, on front
of chest.
Ditto
. Tetanus, 6th July;
confined to left
side, not severe.
Recovered after a
month.
In both these experiments.
quinine was commenced
same day as spores, and
tetanus set in on the
fourth day.
4
Ditto
.
Ditto
.
Ditto
. Same as experiment
No. 3.
5
Ditto
. 3rd to 6th
July, on front
of chest.
Ditto
. Tetanus, 6th July;
severe but confined
to left side. Re-
covered after a
month.
In both these experiments
quinine was commenced
one day after spores, and
tetanus set in on the
fourth day.
6
Ditto
.
Ditto
Ditto
. Same as experiment
No. 5, but disease
less severe.
7 and 8 (Con-
trols of
sp o res
used.)
Ditto
.
Nil.
Nil.
Remained well
.
Both controls remained
well.
9 (Control of
cu1ture
from which
spores
were pre-
pared.)
0.5 c.c. teta-
nus culture,
2nd July.
Nil.
Nil.
Tetanus, 3rd July;
severe and acute.
Died 4th July.
Symptoms local at outset
but rapidly became general.
In experiments 1 and 2 in which quinine was commenced one day before spores were given, tetanus set in one
day earlier than in the other four experiments, in two of which quinine was given the same day as spores, and in
the other two the day after spores.
THE 6TH SERIES OF EXPERIMENTS.
In this series, two guinea pigs were inoculated with "washed tetanus spores,"
and immediately afterwards exposed to a temperature of about 38ºF. in a cold
chamber.
In both cases the spores were injected into the muscles of the right hind leg,
and one of them had at the same time ½ gr. quinine, given subcutaneously on the
front of the chest. The one which got spores only, developed tetanus on the third
day; and the one which got ½ gr. quinine in addition to spores developed tetanus
on the second day, so that the quinine probably hastened the onset of tetanus by
one day.