NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
16
I.—In Persia.
The medium used was ordinary nutrient broth, which was inoculated
from non-ulcerated lepromata of a Persian leper. The tubes were kept at
room temperature. During the period of incubation the broth remained
absolutely clear, and a very small deposit of the transferred material could
be seen at the bottom of the tubes. After eight weeks' incubation small puff-
ball growths appeared at the bottom of each tube, and gradually multiplied,
growing upwards and adhering to the side of the tube.
On microscopical examination these growths were shown, after staining
by Ziehl-Neilsen's method, to consist of a non-acid-fast streptothrix, with
masses of acid-fast bacilli, lying amongst the meshes of the streptothrix. The
first sub-culture showed the same non-acid-fast streptothrix, but the acid-fast
bacilli were fewer in number, and from the third sub-culture onwards no
acid-fast bacilli occur.
II.—In Bombay.
The same streptothrix, producing acid-fast nests has also been obtained
three times from a Goanese leper. In these experiments the tubes were
incubated at 37°C.
(i) In the first case six tubes of Potato broth were inoculated from
different lepromata of said leper. Five tubes sho wed no growth. In one,
however, at the end of ten days, there appeared a very small puff-ball growth
in the bottom of the tube, the supernatant fluid remaining clear. Growth
slowly increased, the colonies climbing up the side of the test-tube, to which
they adhered.
Microscopically.—The growth was a non-acid fast streptothrix producing
acid-fast nests. First sub-culture was similar, but acid-fast organisms were
fewer and from third sub-culture no acid-fasts have been -found.
(ii) From a Rost's medium tube of the "Bacillary" growth to be described
in Section B, I inoculated a large flask of Potato broth. The Rost's medium
tube contained acid-fast lepra bacilli, growing from the same Goanese leper.
After ten days' incubation the flask of Potato broth showed one very small
puff-ball. Growth increased very slowly. It was exactly similar to that
described in the preceding paragraph. Microscopically, also, it was the same
and its subsequent growth was similar.
(iii) From a Rost's medium tube, in which the "Bacillary" growth de
scribed in Section (b), had been growing for about a week, I made inoculations
into six more tubes of Rost's medium. Nothing occurred in five tubes. In
the sixth, after twenty-five days' incubation, four small puff-balls were observed
17
at the bottom of the tube. One removed for examination showed it to be
similar microscopically to those described above. This growth, while similar
microscopically to those grown in ordinary broth, and Potato broth, differed
for a long time, in that it would only grow with the greatest difficulty. After
much perseverance however it has gradually approximated culturally to the
growth obtained in ordinary broth and Potato broth. While growing with
great difficulty on agar media, it was of an orange-red tint. As its growth
became more exuberant it has gradually lost this colouring, and now it shows
the whitish yellow of the other growths, subcultured from the broth strepto-
triceæ.
III.—Growth characteristics of the Streptothrix leproides after initial
culture.
(i) Ordinary Broth.—Itg r o w s r e a d i l y f r o m t h e s e c o n d c l a y a s a f l o c c u l e n t
membrane or puff-ball growth at the bottom of the tube. The medium
remains clear.
(ii) Ordinary Agar.—It grows readily, There is not much surface
growth. The organism digs its way into the agar medium, and the growth on
the surface seems to get less and to be covered with a chalky powder.
(iii) On Glycerine and Glucose Agar.—It grows readily. Whereas in
ordinary agar it digs into the medium everywhere, on Glycerine and Glucose
agar it grows more luxuriantly as a definite membrane on the surface of the
slope and the edges of the growth only grow into the medium.
(iv) Maltose. —Negative.
(v) On. unskimmecl milk at 37°C it grows, after about 10-14 days as a very
definite coherent membrane on the surface of the cream. This membrane
varies in colour according to the medium from which the sub-culture is made
and owing also to variations in the medium, temperature, etc., etc., of which
I am at present ignorant. The full-grown membrane, when subcultured from
Potato broth, is a membrane very like that described by Deycke for his
streptothrix, but the colour is more orange-yellow than orange-red. In the
early part of its growth the membrane is all a non-acid-fast streptothrix.
After about four weeks' growth some parts of it become very strongly acid-fast
and after 6-8 weeks' growth it may break down into bacillary fragments, very
similar morphologically to those obtained in Rost's medium and to be de-
scribed. in Section (B). These bacilli may be acid-fast or non-acid-fast. The
streptothrix is distinctly gram-positive.
(b)—" Bacillus " Leprcæ.—Ih a v e h e a d e d t h i s " B a c i l l u s " L e p r æ t o
separate it distinctly from the more definite streptothrix growth. As stated
18
above, there are reasons for considering it as an altered form of the previously
mentioned streptothrix.
For this series of experiments, I used either Major Rost's original medium,
or an imitation prepared for me at Parel Laboratory. The medium prepared
at Parel was as follows :—
(i) Lemco broth, without the addition of salt or peptone
.
250 c.c.
(ii) Distilled water
.
.
.
.
.
.
.
250 c.c.
(iii) Milk .
.
.
.
.
.
.
.
.
5 0 c . c .
The above differs from Major Rost's medium, in that the distilled water
replaces an equal quantity of the Ngapee (rotten fish) distillate. On analysis
by the Chemical Analyser to the Government of Bombay, it was declared to
be "practically chloride-free."
This series of successful results numbers seventeen, divided between
Bushire (in Persia) and Bombay, and I would emphasise that in each case
where I have got the streptothrix growth, I have also in Rost's medium got the
bacillary form of growth. Further, in two instances recorded above, the
streptothrix form of growth with acid-fast nests of bacilli, has grown out of a
tube of the bacillary form of growth. All these inoculations were made from
non-ulcerated lepromata, and each experiment was controlled with ordinary
media. No growth occurred in the controls of these seventeen experiments.
After inoculation from the non-ulcerated lepromata all tubes were kept at
37°C. After two to three days it was quite evident that there was a multipli-
cation of acid-fast bacilli going on. Positive results are only obtained by
pipetting up from the lowest part of the tube a loopful from half way down the
medium generally shows nothing.
After about seven days' incubation, a small flocculent membrane can be
made out at the bottom of each tube. It is at first very small and is with
difficulty distinguished from the sediment of the medium. This small mem-
brane occurred in all cases. In two cases it was removed and preparations
stained by Ziehl-Neilsen's method were prepared. These showed the membranes
to consist of masses of acid-fast bacilli lying in a zooglœa substance. Fur-
ther examination showed the basis of the growth to be a streptothrix varying
in its acid-fast properties in different parts of the preparation. In this way
growth proceeded very slowly, controls showing that there was nothing present
capable of growing on the ordinary media. At the end of about two months'
incubation, a low power field showed distinct masses of red, scattered about the
field, which under the high power were seen to be made up of acid-fast bacilli.
Attempts were made frequently to obtain a growth on solid media, but for
long without success. After about 2½ mouths' incubation, in tubes of liquid
19
media which showed the best growth, it was noticed that, in addition to the
acid-fast organisms, a non-acid-fast organism, similar in shape to the acid-fasts,
began to appear. This non-acid-fast form is essentially a " diphtheroid "
organism, and, whenever growth has taken a definite move forward, this non-
acid-fast diphtheroid organism has appeared. This non-acid-fast form appeared
too frequently in the liquid cultures for us to consider it as an accidental
contamination, and when we succeeded in obtaining a growth on solid media
we were confirmed in this opinion. After 2½ months' incubation in Rost's
medium from some of the strongest growths, we obtained with difficulty a
growth on Lemco glucose agar (without the addition of NaCl or peptone).
After 8-10 days' incubation on the agar, it was thought that there were signs
of some growth ; this was subcultured and a more definite growth was obtained.
The growth was very weak and consisted of small separate dew drop colonies.
Microscopically these consisted of the same acid-fast and non-acid-fast bacilli
as were seen in the liquid culture.* By subsequent sub-culture in solid and
liquid media the growth has been gradually strengthened until now we have
obtained on solid media a strong growth of a yellow sticky character, which
is similar to Major Rost's most typical growth in its strong acid-fastness ; its
pleomorphism, its yellow colour on solid media and in its power of producing
a severe reaction in leper patients on whom it was tried. It differs in one
important point—whereas Major Rost's growth is now dry and wrinkled, my
growth is very sticky and greasy. This need not, however, be an essential
difference, as Major Rost refers to the change of character of his growth after
frequent sub-culture, and my own experience with one of his organisms
is that of a sticky growth which may be changed by frequent culture into a
dry, wrinkled form of growth.
On staining by the Ziehl-Neilsen method, one sees in a fairly typical
culture of the original weak growth (i) acid-fast lepra bacilli, (ii) similar non-
-acid-fast dotted bacilli, which take the counterstain very faintly, and (iii) other
" involution" forms, such as swollen bacilli, filaments with terminal clubs,
coccoid forms, etc., which take the counterstain strongly.
With Gram's stain it is seen that the elements mentioned under (iii) are
strongly gram-positive, while the others seem to vary in their staining reaction.
Regarding the final strongly acid-fast growth it is more difficult to give a
word picture of what is seen microscopically. What one sees depends on the
* This weak growth has been continued up to the present time (30th January 1911) solely on agar and
retains its original characteristics. The regularity with which the acid-fast bacilli recur in cultures and are
found in single colonies disposes of the criticism made against the non-acid-fast streptothrix grown in broth, viz.,
that the acid-fast nests of bacilli found in the broth growths were those originally transferred from the leper,
which acid-fast bacilli had become accidentally entangled in the meshes of the contaminating streptothrix.
20
manner in which the streptothrix happens to be dividing at the moment. One
may see acid-fast bacilli or acid-fast cocci, and in the groundwork one can
make out at times parts of the non-acid fast streptothrix forms, which are the
basis of the whole growth and in which the acid-fast forms develop.
(c) Are the two organisms identical ? —Without being able to definitely
settle the question by growing one into the other at will, we consider that we
have good grounds for supposing them to be the same organism, altered by
environment.
(i) The " Bacillus " lepræ has been grown from all five cases. The
"bacillus " is almost certainly a phase of the life-history of a
streptothrix, as evidenced by its growth in liquid media and by the
forms seen in growth on solid media.
(ii) Both the streptothrix and the " Bacillus " were got comparatively
frequently from two cases.
(iii) The streptothrix growth on milk breaks down into bacillary elements,
both acid-fast and non-acid-fast, almost identical with those got by
growing the " Bacillus " in liquid or on solid media.
(iv) The streptothrix has twice been grown from Rost's medium in which
the bacillary form of the organism was growing.
(v) Deycke has grown a streptothrix which also breaks down into acid-
fast bacilli.
(vi) Many observers have grown a non-acid-fast-diphtheroid organism.
Both Major Rost's organism and mine are in their non-acid-
fast stage, diphtheroid organisms. They are also both certainly
streptotriceæ.
(vii) Both forms when made into a vaccine cause both general and local
reaction in lepers. Controls on non-lepers point to the specific
nature of the organisms.
(viii) Inoculation of both organisms into monkeys, rabbits, guinea-pigs.
and rats has been so far negative.
(ix) The streptothrix " leproides" when injected into lepers in its non-
acid-fast form produces no reaction. It is only when a certain
amount of acid-fastness has been developed that it produces a
reaction.
(B) DISCUSSION OF RESULTS.
(a) Leprosy has been considered since the discovery of Hansen's bacillus
a fission fungus disease, and indeed, it is so considered at the present time by
most people.
21
To meet criticism of our work, based on this assumption, it will be we
to clear the ground at once, and to point out that, although we find the lepra
organism parasitically as an acid-fast bacillus, it is not therefore necessarily a
fission fungus. It is, indeed, quite possible that it belongs to the mould fungi.
It is impossible for me here to go into a general discussion of the Streptotriceæ
and their varying forms. I would especially refer those intorested to the
Milroy lectures of 1910 by Foulerton. It will be seen from a study of these
most interesting lectures that it is quite possible for a disease to be caused
by a streptothrix, and yet the obvious parasitic form found may be always a
bacillus, or some of her broken down streptothrix form.
I would also refer to the following remarks of Unna as showing that he
evidently has for long been considering possibilities apart from the acid-
fast bacillus. In a paper printed in the "Transactions of the Bombay Medical
Congress," 1909, he says, "as a matter of fact the leprosy organism includes a
large and varied series of forms, which not only include the known bacilli and
globi, but also others, different in morphology and staining reactions, and
transitional forms, which are inseparable from the growth of the leprosy
bacilli in the skin. I will not enter further into the peculiar morphological
shapes, coccus-like granules and granular threads (coccothrix) which were found
in my laboratory by Lutz (1885) and studied more closely by me and my pupil
Spiegel, etc., etc." It is also interesting to note that Hansen himself in his
early attempts to grow the organism, succeeded in getting a mycelial growth,
which, however, later he admitted had nothing to do with the lepra organism.
It is possible that with a knowledge of more recent work lie may be inclined
to change his opinion.
Again, is the leprosy bacillus always acid-fast?
I quote from Allbutt and Rolleston's "System of Medicine" to show that
it is, quite possibly, not always acid-fast. Dr. Abraham, the author of the
article, says: "Herman has also shown that in young or recent nodules, similar
masses of bacilli exist, which do not retain the red stain after immersion in
weak acid, but are easily, subsequently, coloured with methylene blue. If, as
h e s u p p o s e s , i t i s o n l y t h e o l d e r ( f u c h s i n s t a i n e d ) b a c i l l i , t h a t a r e r e s i s t a n t t o
the acid treatment, we may have some explanation of the general failure to
recognise leprosy bacilli in the attempted cultivations, as will be shown further
on."
And, lastly, I would like to draw attention to the fact that many workers,
whose technique is beyond question, although they have failed to grow an
acid-fast lepra bacillus, have described, all of them, the growth of a non-acid-
fast diphtheroid bacillus. Amongst those who describe the growth of a non-
22
acid-fast diphtheroid bacillus are Babes, E. Levy, Czaplewsky, Spronk, Kedrow-
sky and Shiga.* Dean quotes Babes as having grown this non-acid-fast diph-
theroid bacillus from twelve cases of leprosy, and a similar non-acid-fast diph-
-theroid bacillus has been grown from the leprosy-like disease occurring in rats.
Babes evidently thought that his organism was very possibly the lepra bacillus.
He said that although it was possibly the lepra organism in a non-acid-fast
state, one could not consider it seriously unless
(1) it was possible to grow it into the acid-fast state ;
(2) it would produce a tuberculin-like reaction in lepers ; or
(3) unless it could produce leprosy in animals.
The interest of this question of a "diphtheroid bacillus " and in Babes' re-
marks lies in the following facts :
—
(1) Both Major Rost's and my organism are in their non-acid-fast states
diphtheroid bacilli and certain growths tend to lose their acid-fastness
and to become permanently non-acid-fast. With one of Major Rost's
species, this organism definitely regains its acid-fastness on being grown
in a fatty medium like milk.
(2) Both Major Rost's and my organism are capable of producing tuber-
culin-like reactions. Control injections on non-lepers point to the
specific nature of the vaccines.
(3) Major Rost has in one monkey produced a nodular disease, similar
to leprosy, and the nodule contained acid-fast bacilli.
(b) I have during the last two months been experimenting with a vaccine
made from a two months' old streptothrix membrane grown on milk. This
membrane is dissolved in olive oil. In the cases on whom it has been used it
has produced, in appropriate doses, marked general reaction and also local reac-
tion in the leprous lesions. These reactions have been followed by improve-
ment in the leprous condition. Two anæsthetic cases treated with the vaccine
have shown a rapid return of sensation in the affected areas.
A series of control experiments on non-lepers were carried out to exclude the
olive oil alone, and then the vaccine. Five non-lepers were injected twice at
weekly intervals with olive oil (equal in amount to the vaccine) and 5 non-
lepers were injected twice with the vaccine. Those injected with olive
oil were not affected in any way by the injection. Those injected with the
*It is particularly interesting to refer to Shiga's description (Transactions, Bombay Medical Congress, 1909)
of the diphtheroid organism isolated by him. It will be seen that by inoculating fresh leprous tubercle on to
potato glycerine agar with human serum, he obtained, after four weeks' incubation, the growth of a diphtheroid
organism which was not acid-fast, but amongst the non-acid-fast organisms were seen acid-fast lepra-like
bacilli. He thought, as did we, of the possibility of symbiosis, but further work showed that it was a pure
culture. In his case, as in ours, the agglutination test was not certain, as leper serum and healthy serum reacted
about the same.
23
vaccine suffered a certain amount at the site of injection for about 24 hour
and two had slight fever. None of the controls, however, in any way
approached the severe reaction which I have obtained in cases of leprosy
I n t h e a n æs t h e t i c c a s e s , wh e r e o n e wo u l d e x p e c t t h e n u mb e r s o f b a c i l l i t o
be less than in pronounced tubercular cases, reaction has been very slight,—
hardly more than in the controls. In definite tubercular cases reaction
has been severe, and judging by my results on five cases—three tubercular and
two anæsthetic,—the reaction appears to be proportionate to the severity of the
disease.*
(c) In a subject like leprosy, it is from every point of view necessary to
go slowly and not to make definite claims until they can be proved up to
the hilt. I am well aware that our present work does not reach this standard.
I think, however, that the results merit favourable consideration, and they
are put forward at this juncture in the hope that others interested in leprosy
may be induced to attack the subject along the same lines.
*Since writing the above in September 1910, we have discarded the vaccine made by dissolving the
streptothrix membrane in olive oil.
For some time we experimented with a vaccine made by scraping the acid-fast growth off agar tubes ;
drying in vacuo ; powdering in a mortar ; and then dissolving the material in normal salt so ation. This gave
rise to similar reactions, both general and local, to those produced by the olive oil vaccine. The results were
not, however, regular enough to be satisfactory. After consultation with Major Rost, we decided to try the
injection of a six weeks' old. bouillon culture of the organism. These experiments have only been in operation
for the last three weeks and one would not be justified in saying much about them, but there can be no doubt
that the bouillon vaccine is much more potent in so far as producing a reaction is concerned. The definite charac-
ter, moreover, of the local reaction in the leprous lesions, in many cases, supports the view that we are dealing
with a specific remedy (T. S. B. W., 30.1-11.)
(NEW SER I ES)
N®0 42.
SCIENTIFIC MEMOIRS
BY
OFFICERS OF THE MEDICAL AND SANITARY DEPARTMENTS
OF THE
GOVERNMENT OF INDIA.
PART 1.
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.
PART II.
The Cultivation of the Leprosy Bacillus
BY
CAPTAIN T. S. B. WILLIAMS, M.B., I.M.S.
(EDITED BY THE DIRECTOR-GENERAL, INDIAN MEDICAL SERVICE.)
ISSUED UNDER THE AUTIIORITY OF THE GOVERNMENT OF INDIA BY THE SANITARY COMMISSIONER
WITH THE GOVERNMENT OF INDIA, SIMLA..
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1911
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No. 56.
SCIENTIFIC MEMOIRS
BY
OFFICERS OF THE MEDICAL AND SANITARY DEPARTMENTS
OF THE
GOVERNMENT OF INDIA
M A L A R I A I N T H E A N D A M A N S
BY
MAJOR S. R. CHRISTOPHERS, M.B., I.M.S.
Assistant to the Director, Central Research Institute, Kasauli
ISSUED UNDER THE AUTHORITY OF THE GOVERNMENT OF INDIA BY THE
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1912
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No. 56.
SCIENTIFIC MEMOIRS
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MALARIA IN THE ANDAMANS
BY
MAJOR S. R. CHRISTOPHERS, M.B., I.M.S.
Assistant to the Director, Central Research Institute, Kasauli
ISSUED UNDER THE AUTHORITY OF THE GOVERNMENT OF INDIA BY THE
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1912