NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
4
leper nodules. Both in our results obtained here and also in those obtained
by Major Rost in Rangoon, the growth appears in about three days, at the
bottom of the tube. Smears made from the growth at the bottom of the tube
show acid-fast bacilli, similar morphologically to those seen in smears made
from leprous nodules. Whether these are the actual bacilli of leprosy or not,
it is a noteworthy fact that no growth is obtained under similar conditions in
the controls on ordinary media. The organism is highly pleomorphic and
likewise varies in its staining reactions, all stages between complete acid-fast-
ness and the reverse being seen in a single preparation. These variations in
form and staining reaction are quite compatible with the life-history of the
streptotriceæ as given by Foulerton, and with its being a pure cultivation.
The above remarks have reference especially to the bacillary form of growth
as isolated in Rangoon and here. As will be seen by a reference to Williams'
paper a very definite streptothrix has been grown by him in ordinary media
from two cases in Persia and Bombay. In our examination of Rost's organism,
we also convinced ourselves that one species at least produced at one stage a
definite mycelial growth and even those, where a mycelial stage has not been
seen, are certainly streptotriceæ. Further Deycke has grown a streptothrix
from cases of leprosy which is very similar to that produced by Williams.
A consideration of all these points together with the evidence produced in
Williams' paper as to the streptothrix and bacillary form being but phases
of the same organism, gives one ground for seriously considering anew the
whole question of leprosy, and it seems to me that there is now a considerable
amount of weight to be attached to the view that leprosy is very probably after
all a streptothrix disease.
4. From a study of the papers of Rost and Williams and from a knowledge
of the work done in this Laboratory, I am of opinion that the organisms
found by Rost and Williams are probably identical. It will be seen from a
perusal of their papers that in Rost's special medium their results are similar
and the ultimate result has been the production of acid-fast organisms by both
which are similar in their pleomorphism, acid-fastness, being streptotriceæ,
and in their power to produce marked reaction in lepers. The only difference
at present is that Williams' growth is sticky, while Rost's growth is dry.
From Major Rost's paper, it would seem that his growth was originally sticky,
but has altered by prolonged sub-culture.* It is also very interesting to note
that Williams has grown several times a streptothrix which is very like that
of Deycke, as recorded in his published results, and its growth on milk is
* Since the above was written, Captain Williams has been to Rangoon and has demonstrated that, under
certain conditions, Major Rost's organism loses its wrinkled character and becomes sticky ; whilst the organism
isolated by himself is capable of growing as a wrinkled membrane.—(EDITOR)
5
strikingly similar to the growth of Professor Deycke's organism. Williams
is of opinion, and I am somewhat inclined to agree with him, that his work
links up that of Deycke and Major Rost, and that his two organisms are,
really, one and the same organism, which has been very decidedly altered by
its environment, and by the original medium in which it was grown. The
details given in Williams' paper, and the fact that both his organisms produce
an apparently specific reaction in lepers, such as is produced by Deycke's
and Rost's organisms, would seem to be strong evidence in favour of this view.
5. In conclusion, I would like shortly to discuss the possibilities of success-
ful treatment of lepers by a vaccine from these organisms. Major Rost
describes in his paper very satisfactory results, some of which, I understand,
you have yourself seen when you were in Rangoon. We have here for the
last few months been using a vaccine made first of all from the streptothrix
grown on milk, and later from the bacillary form of the organism grown from
Rost's medium. Our results are most encouraging and, in our opinion, tend
to confirm Major Rost's work.
C
PART I.
THE CULTIVATION OF THE BACILLUS OF LEPROSY AND THE
TREATMENT OF CASES BY MEANS OF A VACCINE
PREPARED FROM THE CULTIVATIONS.
BY
MAJOR E. R. ROST, I.M.S.
A FTER research into the Bacteriology of Leprosy extending over a period of
seven years, I succeeded in 1909, with the assistance of my Clinical
Assistant, Mr. Bansi Lal, in isolating from three cases of Leprosy an acid-fast
bacillus, which I was able to subculture in pure growths through successive
generations, and which possessed certain peculiar characteristics, resemblnig
morphologically the bacillus of Leprosy.
The first case from which I succeeded in isolating the bacillus was one
of long standing nodular Leprosy in a Chinaman, who periodically developed
large blisters, which appeared spontaneously on anæsthetic areas without being
caused by burns or irritants. Microscopic examination of the blister fluid
showed in some instances a few acid-fast bacteria. The blister fluid drawn off
into pipettes and incubated at room temperature for five days showed a marked
increase in the number of acid-fast bacteria, and controls taken from the
pipettes in ordinary media remained sterile. The pipettes showed white flakes,
and, on microscopic examination, acid-fast bacteria were found massed together
in parallel arrangement.
The medium used consisted of 250 c.c. of distilled volatile alkaloid of rotten
fish, 250 c.c. of weak Lemco broth without salt or peptone and 50 c.c. of milk
and was prepared in the following way :—
A bottle containing (Ngapee)* Burmese preserved fish is provided with
a rubber stopper perforated with 2 holes through which steam could enter down
a glass tube to the bottom of the bottle and pass over the contents and issue
through a glass tube at the top ; this bottle is placed in an autoclave and the
Outlet tube from the bottle led out through the autoclave into a condenser.
* Major Rost, perhaps, attaches too much importance to the Ngapee distillate. Williams has succeeded in
growing the Leprosy bacillus in a medium in which distilled water is substituted for the Ngapee distillate.—ED.
8
Steam generated in the autoclave passes over the heated fish (which has a little
caustic soda added in order to bring out the volatile alkaloids more readily) and
passes out of the autoclave into the condenser and is collected in a flask.
The medium is then mixed with previously prepared and autoclaved.
Lemco broth and milk in the above proportions, placed in tubes and auto-
claved.
The medium was controlled on ordinary agar and broth at the time of the
experiments. Tubes of this medium were inoculated from the blister fluid
which had been previously left in pipettes at room temperature for five days.
These pipettes now contained large masses of acid-fast bacteria, resembling
morphologically that of Leprosy, whereas previously only a few were found in
the blister fluid.
Three days later a slight stringy growth was visible at the bottom of the
tube and films showed a fairly large number of acid-fast bacteria, massed to-
gether in parallel arrangement.
It was then found that a feebly acid-fast bacillus grew on nutrient agar
and broth (without salt or peptone) in subcultures, and that the acid-fastness
of the bacillus was regained by growing in milk, and that the degree of acid-
fastness could be varied according to the fatty nature of the medium. Plating
experiments were then carried out and in these five loopfuls of the culture were
stirred in the nutrient agar (without salt or peptone) cooled down to 40°C.
Three loopfuls were taken from this tube and removed to a second tube of
agar medium which was poured into a Petri dish. Three days later the plates
showed discrete colonies about the size of a pin's head, opaque, orange-red,
raised with the centre, humped and moist to the naked eye.
Examined with a lens these colonies were circular, opaque, warm orange-
red coloured, with regular unbroken margins. One of these colonies was
inoculated into a tube of the primary medium and this formed the basis of
experiments with this first cultivation.
By continuing the sub-culture and by occasional plating a slight variation
was obtained ; two slightly differing cultures being secured, which by successive
subculture again became identical in appearance.
The second successful isolation, producing a bacillus the cultural charac-
teristics of which are practically identical with the first and differ very slightly
only in rate of growth and moisture of colonies, was obtained from a European*
suffering from nodular Leprosy. The fluid exudation from a cut nodule was
used in this case, inoculation into the special medium being direct. Incu-
bation at room temperature 30°C. in this case took longer than in the former
case, a visible growth appearing after three weeks.
* This case is now practically cured by the use of a vaccine prepared from these cultivations.
9
This appeared as a slightly yellowish red growth on the surface of the
medium. Subculture on ordinary media without fat showed the same loss
of acid-fastness, which could be varied according to the fatty nature of the
medium.
This culture appeared to grow more slowly and to be slightly more sticky
than the first isolation. It has gradually altered in characteristics, and, after 18
months of successive subculture has become more like the culture first isolated.
In two other cases the same organism was isolated ; but the cultures being
found on plating to be impure, these cultivations were discontinued.
In November 1909, by adopting a medium similar to that used with the
first pure culture, a growth was obtained from a nodular case of Leprosy. This
was a Burman lad, aged 18, Kone Sein, who showed on his phalanges and
face, a number of hard spotty nodules not larger than a pea, pyramidal shaped,
glazed and shining. The method employed was to reflect the skin with
aseptic precautions, over one of these recent nodules and squeeze out the
fluid ; inoculate it into the special medium and control also in ordinary media.
The tubes were incubated at 30ºC. The controls remained sterile.
The special media tubes showed a number of acid-fast clumps similar to
those found in the leper juice after fourteen days. Later on, these acid-fast
masses began to diminish and after the third week required a prolonged search
to find them. During the fourth week a steady increase was observed in these
acid-fast clumps and several acid-fast bacilli were found to occur singly as
though the organism was multiplying, but it was only at the end of the fourth
week that the whole culture medium was observed to be impregnated with
acid-fast bacteria.
After the fifth week a thin pellicle was found on the surface, this con-
sisted of small, greyish, coarse granular flakes of a crumbling consistence.
From the original culture, plates were inoculated and in these the colonies
appeared after a week and showed similar characteristics to those previously
isolated, with the exception that the rate of growth was in the first instance
slower, the colour a lighter yellow and the acid-fastness constant whether
grown on fatty media or not : otherwise the subcultures from this case, in
appearance and peculiarities of growth as detailed below, are identical with
the other previously isolated cultures.
First and second successful Isolation O . L. O.
and T. L. O.—H. O. and H. T.
Third successful Isolation.
Kone Sein.
1. Grows best on pure milk ; on milk 50, Lemco-broth 250 (without
salt or peptone) and distilled rotten fish medium 250.
. . . . . .
2. Primary isolation growth has not been obtained by any other
medium yet discovered.
. . . . . .
10
First and second successful Isolation O. L. O.
and T. L. O.—H. O. and H. T.
Third successful Isolation
Kone Sein.
3. It grows on primary cultivation slowly . . . . .
......
4. Can be subcultured direct from primary growth on to the other
media.
Does not grow on first subculture
in ordinary media but does so after
successive subculture in special
medium.
5. On all other media, except fish broth, it appears to degenerate
rapidly into attenuated forms of bacilli and becoming feebly acid-fast,
strong broth and peptone and salt enhancing this degeneration.
......
6. It grows on pure starch and potato-glycerine but not on pure
albumen.
......
7. Its acid-fastness varies with the fatty nature of the media used :
on milk it is acid-fast and on fish broth very much so.
Acid-fastness constant on all media.
33% HNO3 ,
—2 mins.
Milk.
Fish broth.
25% H2SO4
—½ hour.
25% H2SO4 . . .
5 mins ±
—2½, mins.
12½% H2SO4 .
.
.
.
20 „ ±
—10 „
5% H2SO4 .
.
½ hour ±
—½ hour
8. It stains readily in the cold with simple aqueous aniline dyes and
is gram positive.
.......
9. It is non-motile.
......
10. Grows best at 30°C. After a few generations from the original
culture it grows in 48 hours.
......
11. It is exceedingly pleomorphic, appearing in different forms at
different periods of its growth. After 48 hours' growth its appearance
is as in the nodules of a Leper. Later on, or in unfavourable media
and temperature, degenerate forms are found which double or treble
their usual length with a monilliform arrangement and lase their acid
fastness. These break-down after a few days into clumps of small
acid-fast coccoid forms. The coccoid forms are more evident in some
cultures than in others.
In Kone Sein culture the thread
like forms have not been found to
occur.
I.
II.
III.
First successful Isolation.
Second successful Isolation.
Third successful Isolation.
12. In old cultures an orange-red colour is seen at the bottom of the
tubes. In milk it grows first as a layer just under the cream, In broth
it grows first at the bottom as a thin whitish loose deposit rendered
visible by shaking the tube. The upper portion of the broth remaining
clear for the first two days and then pinkish flakes forming on the
surface which show the disintegrating forms.
It grows luxuriantly on pure milk
as a layer under the cream, but the
orange-red colour is absent. In old
cultures the colour has a tinge of
yellow but has not become red as
the two previous isolations.
On agar its growth is slower and
it assumes a more crinkled appear
ance. On glycerinated agar the
growth is more luxuriant. In broth
it falls as a powdery deposit at the
bottom of the tubes while the
medium is not clouded, in glyceri
nated broth there is no superficial
growth except after prolonged culti
vation.
11
13. Films are best prepared by filtering a culture through filter paper ; thus removing fatty masses and better
specimens are thus obtained.
14.The characters of the cultures have slightly changed over a period of eighteen months, the different
isolations from the three different cases tending to become more exactly alike, except the Kone Sein culture
which does not vary its acid-fastness.
15.The following test media showed in each case:—
Growth.
Acid.
Gas.
REMARKS.
Glucose Litmus .
.
+
—
—
Colour of litmus taken up by the
growth after some time, leaving clear
fluid, but colour of litmus not chang-
ed.
Mannite „
.
.
+
—
—
Dulcite „
.
.
+
—
—
Lactose „
.
.
+
—
—
Bile Salt „
.
.
—
—
—
16.After exposure at 80ºC. for 15 minutes no subculture was obtained.
17.1 in 500 bile salt prevents growth in all cases, sheep's bile 1 per cent. prevents growth.
18.The indol reaction is negative.
It will be seen from the above tables that the only differences between the
two first isolated cultures and the third or Kone Sein culture are colour, rate
of first growth, extent of acid-fastness and the absence of long thread-like
forms in the Kone Sein cultures. Otherwise the characteristics of the three
are the same, their pleomorphism being so marked and peculiar as to lead one
to imagine that the culture is not pure; the varying acid-fastness in the first
two isolated cultures tending, on first examination, to lead one still further
astray.
Inoculation Experiments.—Injections of culture alone and mixed with
butter were made into guinea-pigs, white rats, and rabbits, subcutaneously,
by tattooing, by feeding and intra-peritoneally without result.
A monkey repeatedly injected with culture, developed clinical signs of the
disease and nodules appeared in which were found typical lepra bacilli, but I
failed to produce a pure cultivation. The history of this case is as follows:
—
This monkey was first injected intra-peritoneally in October 1909, the
injections being continued weekly, and the method of inoculation being varied
between intra-peritoneal injections, tattooing and applications of poultices of
culture and subcutaneous inoculations.
In December 1909, he began to develop exacerbations of temperature,
which varied between 100 and 103, and nodules were noticed over the super-
ciliary ridges; on the cheeks and inner aspect of the left ankle; and on the
dorsum of the left foot.
At the end of December, the hard mass on the ankle was found to
contain pus and this, when examined under the microscope, contained several
clumps of acid-fast bacteria, resembling in appearance Lepra bacilli.
12
Acid-fast bacilli in parallel arrangement were found situated inside cells
in smears prepared from the pus.
The injections were then stopped.
In February 1910, the nodules on the superciliary ridges were examined
microscopically, the monkey being chloroformed and a nodule being excised
partly for the purpose of obtaining a cultivation and partly for microscopic
examination. The microscopic smears from this nodule showed numerous
masses of acid-fast bacillary groups in parallel arrangements, resembling exactly
specimens obtained from the human subject.
Cultivation gave negative results.
The monkey being a small animal the nodular lesions were of course
small and photography failed to reproduce what was more easily visible to the
naked eye. The superciliary ridges were raised and thickened and had a glisten-
ing red appearance. The nodules on the cheeks appeared to subside in
February as did the nodules on the superciliary ridge, but the fever
continued, the monkey gradually wasting away. Examination of the blood
showed nothing.
In April 1910, the monkey died apparently from inanition, and post-
mortem examination revealed no abnormality of the internal organs.
Smears taken from the sub-nodular area of the eye-brows showed some
small groups of acid-fast bacteria in parallel arrangement resembling Lepra
bacilli.
Cultivation from these failed, owing to contamination by skin bacteria,
although great care was taken to sterilize the superficial skin.
Eight other monkeys have been injected with cultures of varying days'
growth, but so far no clinical sign of the disease has been observed.
The Preparation of a Vaccine.—The first vaccine used was prepared from
the subcultures obtained after plating from the first primary isolation.
Bacteria removed from an agar slope culture were shaken up with distilled
water and centrifugalized, the fluid being poured off and fresh distilled water
added, and the deposit shaken up again, and again centrifugalized several times,
so as to wash the culture and remove all external toxins. The deposit of bac-
teria, after final washing and centrifugalising, was dried and weighed and
macerated with 7 per cent. glycerine and distilled water to make up a
percentage solution. It was then placed in tubes and autoclaved, the tubes
being sealed.
The sealed tubes were then placed on a shaking machine for a period ex-
tending over several weeks. Ten minims of 1 in 400 of this vaccine produced
a slight febrile reaction in cases of leprosy, and its therapeutic usefulness has
been very marked.
13
In May 1910 the vaccine was prepared from the Kone Sein culture, and
since this date this culture has been used in the preparation of the vaccine.
Several modifications of preparation of this vaccine were tried previous to
this, but the above mentioned method appeared to produce a vaccine having a
greater clinical value.
Lately, the fatty substance of the bacteria has been extracted by shaking
in ether over a period of several weeks, occasionally drawing off the ether and
adding fresh, filtering and centrifugalizing the deposit and evaporating down
the ether extract until it became of a sticky subsistence, then adding olive
oil to a weighed amount to make up as far as possible a percentage strength.
The therapeutic use of this has been, as far as one can tell, rather more
useful, though at present it is too early to compare the two methods clinically.
The Clinical Use of the Vaccime.—The method of treatment of voluntary
cases at the Leper Asylum, Kemendine, by the use of a vaccine prepared from
these cultures was commenced in March 1909.
Up to January 1910 the autoclaved vaccine prepared in the usual way was
used : after that date the special washed bacillary vaccine, as described above,
has been used. The ethereal extract of bacillary fat has been tried with bene-
ficial result in a few cases, but it is at present too early to report on this
variation.
Of the ten cases in which this treatment has been adopted, two have now
recovered ; two are so much improved that apparently the remnants of the
disease are very slight ; and the remaining six have all improved in a remark-
able manner.
In these cases, with one exception, the injection of vaccine was the only
treatment adopted.
A full clinical report on these cases will be published later on in one of
the medical journals.
15
PART II.
THE CULTIVATION OF THE LEPROSY BACILLUS.
BY
CAPTAIN T. S. B. WILLIAMS, M.B., I.M.S.
During the last six years I have devoted special attention to the leprosy
problem, and this paper contains a résumé of my results up to the present
time. Prior to the beginning of 1909 my results were of an indefinite nature,
and although useful to me, in helping me to judge the value of my later work,
they would be out of place in this paper.
Up to the end of 1908 I had regarded the Bacillus of Leprosy as a
true fission fungus, and had looked for it always as an acid-fast bacillus. My
own and other people's negative results, combined at this time to cause in my
mind a reconsideration of the problem. As a result of my work since the
beginning of 1909, I have grown from five cases of leprosy, under circum-
stances which to my mind preclude contamination, two apparently different
organisms. Two cases were in Persia and three in Bombay. Later work has
tended to the belief that they are really the same organism, altered by environ-
ment. Shortly, I may say that in ordinary broth, I have grown a streptothrix,
somewhat similar to that described by Professor Deycke, while in other special
media, I have grown a "bacillus" very similar to that grown by Major Rost
in Rangoon.
(A) BACTERIOLOGICAL RESULTS.
In all our experiments, it goes without saying, that every attempt has
been made to avoid contamination, and ordinary media have always been used
to control the technique employed, both at the initial transference of material
and subsequently.
As stated above, I have grown two types of organisms : (a) a streptothrix,
and (b) a "bacillus", which is almost certainly the bacillary form of a
streptothrix. It will be more convenient to describe them separately.
(a) —Streptothrix "leproides."—This organism has been grown by me
three times in Persia, from a Persian leper, and three times in Bombay,
from a Goanese leper.
D 2