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SCIENTIFIC MEMOIRS
BY
MEDICAL OFFICERS OF THE ARMY OF INDIA.
BDITBD BT
B. SIMPSON, Esq., M.D.,
8UROBOM-OBNBRAL WITH THB OOVBRNMBNT OP INDIA.
PART I.
1884.
I. — On the relation of Cholera to Schizomycete organisms.
D. D. CunningfuiTfi.
3.— -On the presence of peculiar parasitic organisms in the tissue of a
specimen of Delhi Boil.
D. D. Cunningham.
c
CALCUTTA :
PRINTED BY THE SUPERINTENDENT OF GOVERNMENT PRINTING, INDIA.
1885.
Prioe 2 rupee*.
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On the relation of Cholera to Schizomycete organisms.
Surceon-Major D. D. CUNNINOHAM, mo.,
SPECIAL ASSISTANT TO THE S
The following paper dealing with certain points in the vexed question of the
relation of cholera to schizomycete organisms, and specially of its relation to
so-called " Nomina bacilli," contains an account of the results attained in a.
series of investigations which were conducted in Calcutta during the current
year and mainly during the hot and rainy seasons.
In order so far as possible to avoid laying undue importance on all matter
of a purely controversial nature, attention has been almost entirely confined
to an account of the observations themselves, only occasional and casual re-
ference being made to the data which have been already published by other
observers. This has not been done from any want of recognition of their value
or from any wish to depreciate the work of others, but solely because the state-
ments which have been made in regard to many points are so conflicting and
irreconcilable in their nature that, were any special reference made to any appa.
rent confirmation or contradiction of any of them, an appearance of partisanship
might be imparted to what ought to be a mere statement of results.
The entire series of observations was specially related to the following
points : —
I, — The association of curved schizomycete organisms with materials
of choleraic nature ;
II. — The presence of similar bodies in other media ;
III. — The effects of variations in the nature of the medium in which they
are developed on the curved schizomycetes occurring in associa-
tion with choleraic materials.
Due to the time of year at which the cultivations of choleraic and other
materials were principally carried on — a time during which the conditions of
temperature were such as to preclude the use of common gelatine solid media —
they were all conducted in agar-agar jelly or in fluids of various kinds. The
agar-agar jelly was prepared according to the formula for the common gelatine
Scientific Memoirs by
basis in Professor Johne's account of the methods followed in Dr. Koch's
laboratory during the conduct of the courses of bacteriological teaching carried
out there in reference to the diagnosis of cholera by means of the results of
Cultivations of comma bacilli.' The amount of agar-agar employed was,
however, considerably less than that of the gelatine in the formula, as it was
ascertained that about r5 per cent, was sufficient to secure a basis which would
remain solid in the temperatures to which it was ordinarily exposed. As the
temperature was fairly uniform and constantly high, comparatively little use was
made of the incubator, the cultivations being, as a rule, conducted at the ordi-
nary temperature of the laboratory.
In some cases the substratum was neutralised or rendered faintly alkaline
by means of the addition of solutions of carbonate of soda ; in others its norma]
acidity was reduced by similar means; and in still others it was allowed to
retain its natural reaction.
This substratum served as a basis for plate and tube cultivations, and in
certain of the latter was so far diluted as to cause it to remain permanently
fluid. In other cases acid, neutral or alkaline broth, was employed in tube,
cultivations.
Microscopical preparations of the materials employed in the cultivations
and of the growths connected with them, were made by the usual methods of
preparing dried films on cover- glasses, staining these with different aniline dyes
and washing out the excess of color in spirit. A number of dyes were occa-
sionally employed, but in the majority of instances Spiller's purple and fuchsin
were those which were used. Where sections of tissues were prepared Williams'
freezing microtome was made use of, as it was found that in an atmosphere so
hot and humid as that of Calcutta is during the hot and rainy seasons instru-
ments depending on the action of ether did not give satisfactory results.
The microscopes employed in the examination of specimens consisted of a
Zeiss fitted with an Abbe's condenser and lenses B, C, D, and -^-^ oil immersion,
and one of Beck's "Pathological" microscopes with lenses yV, \, and ^ of
the same makers, and ^\ and ^V oil immersion lenses by Powell and Lealand.
Each of the special subjects of investigation will now be considered in suc-
cession.
I. — The association of curved Schizomycete organisms with materials
of choleraic nature.
A.— Results of Examinations of recent Choleraic Materials.
/. Alvine evacuations.
The materials of this nature which were examined were obtained from the
majority of the patients who were admitted into the wards of the Medical Col-
lege Hospital during the current year as cases of cholera. By far the greater
number of these occurred during the months of March, April, and May, there
' Ueb«r die Koch'schon Rein, ul urea und die Cholera Bacillen.— Von Dr. Albert Johne Leipzig, 1865.
Medical Officers of the Army of India.
having been hardly any choleraic admissions during the latter part of the cold
weather and the normal fall in prevalence of the disease having taken place
towards the close of the hot weather.
The following table shows the total number of cases from which materials
of any kind were obtained during each month from January to September: —
January ........... o
February .......... 3
March ........... g
April . . . . . . . ■ . . .11
May ........... 6
June 3
Ji-ly 3
August ........... I
September .....•....!
37
It is curious to observe how even in such a limited number of cases the
monthly distribution agrees with the data which we possess regarding the gen-
eral normal seasonal prevalence of the disease.
It will be seen that the total number of cases of supposed choleraic nature
from which materials of any kind were obtained was 37. One of them, however,
that occurring in August, was subsequently ascertained not to be of a choleraic
nature, and in regard to one or two of the others, a suspicion that the same was
the case certainly exists. These cases will receive special notice further on.
Samples of fresh alvine evacuations were obtained in 28 cases. In 26 of
these cur\'ed schizomycetes were recognisable. In two of them none could be
detected. In both of these the flocculi which were prepared for examination
appeared to be composed of almost pure mucus containing numerous blood
corpuscles and curiously polynucleate mucus corpuscles but hardly any schi-
zomycete organisms of any kind.
The numbers as well as the characters of the commas present in the
remaining 26 cases varied very greatly. In ten cases they were very abundant,
and in several of these the material approached a pure cultivation in its charac-
ters. In seven of the ten cases the commas were characteristic in appearance
resembling those developed in artificial cultivations. In two they were charac-
terised by their length, slendemess, and slight degree of curvature. In one they
were quite unlike those occurring in any other instance, consisting of very large,
relatively thick, strongly curved bodies. In the remaining 16 cases they were
present in varying but relatively small numbers and of characteristic appearance.
2. Intestinal contents.
Specimens of Iliac mucus were examined in five cases.
In all cases commas were present. In four cases very few were present
lA
Sctenfifc Meinoirs by
and straight bacilli of various kinds abounded. In the specimens derived from
the remaining case the conditions were reversed commas being present in veiy
great numbers and few specimens of any other form being associated with them.
In connection with this subject it may be well to note thai in none of the
cases from whicii these materials were obtained was there any conspicuous signs
of congestion of the intestinal mucous membrane, and that in two of them not
the faintest evidence of any such condition could be detected.
2. Blood.
Specimens of blood were obtained from eight cases.— in four during life and
in the others during the course of post morlem examination. The samples In the
first class of cases were taken from the tips of the fingers which had been pre-
viously carefully cleansed with spirit and solution of corrosive sublimate. The
needles employed for puncture were heated to redness Immediately previous to
being employed and the blood was received In freshly drawn capillary pipettes.
in those cases where the blood was obtained after death the samples were
taken by means of passing the extremities of freshly drawn closed capillary
pipettes through the walls of the right auricle and subsequently breaking of the
points within the cavity.
Numerous preparations were made from the materials thus obtained, but in
no instance could the presence of schizomycete organisms of any kind be de-
tected in them.
B.— Results of Cultivations of Choleraic Materials,
/. Cultivations of alvlne evacuations.
Plate cultivations of this nature were carried out In i8 cases and in 13 of
these colonies of characteristic commas made their appearance. !n the remain-
ing five cases no commas could be detected. In one of these cases the substra-
tum of the cultivation was of acid reaction, in one the material from which the
cultivation was derived was certainly not of truly choleraic nature, and In two
others it appeared to be very probable that the same was the case, the reaction of
the evacuation being of a strongly acid character In one of them, and the micros-
copical features of the other being very exceptional and unlike those ordinarily
present In choleraic materials- In one case only in which there could be no
reasonable doubt as to the choleraic nature of the disease, and In which the cul-
tivation was conducted in an alkaline substratum did commas fail to appear.
The proportions which the colonies of commas bore to those of other forms of
schizomycete organisms varied very greatly in different instances. In some
cultivations only one or two colonies could be detected among numerous others
of different nature ; in other cases they were present in large numbers ; and In
one, at all events, an almost pure cultivation of commas occurred.
Medical Officers of the Army of India.
2. Cultivations of materials obtained from the intestinal cavity after death.
Cultivations of Iliac mucus were carried out in four cases.
In three instances crops of characteristic commas were obtained. In two
of them colonies of commas were developed in large numbers. In the third case
they were very scanty, whilst colonies of minute, straight bacilli abounded.
In one case no commas could be detected. In this the substratum of the
cultivation was of an acid reaction, but this condition was also present in one
of the cultivations which yielded an abundant crop of commas.
J. Cultivations of blood.
Eighteen distinct tube cultivations of blood obtained from the eight cases o(
cholera previously alluded to were carried out, three or four punctures being
made in each tube. In 16 tubes no development of any kind occurred. In the
remaining two, which both belonged to the same case, a growth of what ulti-
mately proved to be Micrococcus luteus took place along the lines of puncture
and gradually spread thence over the surface.
4. Cultivations of other chaleraic materials.
In three cases tube cultivations of juice from the liver and kidney of cases
of cholera were attempted. The material for cultivation was obtained by means
of carefully cleansing the surface of the organs n-ith spirit and solution of cor-
rosive sublimate, introducing the extremity of a freshly drawn, closed capillary
pipette through the cleaned area into the subjacent tissues, and then breaking
the tip and applying pressure around it. In two cases the cultivations were not
at'.ended with any result. In the remaining case the" cultivation of kidney juice
behaved in the same fashion, while schizomycete organisms of various forms ap-
peared in that of liver juice. These, however, were unquestionably of salivary
origin and connected with the fact that strong suction had been applied to the
distal extremity of the pipette in the attempt to obtain a sufficient supply of juice.
Certain points in connection with the cultivations of materials of intestinal
origin call for somewhat closer consideration here than they have met with in
the course of the above general statement of results. These specially relate to
those cases in which no development of commas occurred as the result of cul-
tivations. Taking the entire series of primary plate cultivations of intestinal
materials, it will be seen that they amounted to 22, that a development of
commas occurred in 16, and that in six of them no commas were detected. It
has been already pointed out that in one case the material cultivated was not of
choleraic origin, that in two cases the nature of the material was doubtful, and
that in two the nature of the substratum might be regarded as accounting for
the phenomenon, so that only a single case remains in which an apparently
unequivocal choleraic material cultivated in a seemingly favourable substratum
failed to produce a recognisable crop of commas.
Scientijic Memoirs by
With regard to this latter case, however, I must here draw attention to
three points. In the first place it must be allowed that the failure to detect
commas in the cultivation does not absolutely prove their actual absence from
it. This is due to the well-know-n fact that colonics of commas and other
schizomycete organisms cultivated on agar-agar jelly fail to present the same
constant, well-defined, specific macroscopic characters which they possess in
common gelatine cultivations; so that, although it is in most cases possible to
determine readily that certain colonies are not composed of commas, it is at
the same time impossible macroscopically to distinguish those that are com-
posed of commas from colonies belonging to certain other forms. This being so,
it follows that where commatous colonies are only present in small numbers Jn a
mixed cultivation, they may fail to be detected unless specimens from every
individual colony present be examined. Now in the present instance this course
was not followed ; but, on the contrary, only a very limited number of colonies
were examined due to the cultivation having been carried out at a time when a
press of other work was in hand. In the third place, believing as i do in the
polymorphic nature of the commas associated with cholera, I do not feel con-
vinced that in any case such as this, where only a single primary cultivation was
conducted, the mere absence of recognisable commas satisfactorily demon-
strates the absence of the organism. Unless a succession of cultivations be
carried out under conditions favourable to the appearance of commas, it is
impossible to determine that any material, however free of actual commas, does
not contain potential ones — does not contain forms which under certain condi-
tions would be capable of becoming converted into or of giving origin to
commas. In the present case colonies of minute straight bacilli abounded in
the cultivation, and I do not think it impossible that these were developmentally
related to commas.
The facts in regard to the case in which it was distinctly ascertained that
the materials cultivated were really not of choleraic origin are the following. An
extensive series of cultivations of normal alvine materials had led to the conclu-
sion that the curved schizomycete organisms occurring in them, although often
present in considerable numbers, were not reproduced in artificial cultivations In
neutral or alkaline agar-agar jelly, and that they therein differed from those
occurring in choleraic media. In order to obtain a sharply defined demonstra-
tion of the existence of this difference advantage was taken of the arrival of a
supply of material from the hospital derived from a case, admitted as one of
cholera, to start simultaneous plate cultivations of it and of a healthy alvine
evacuation containing appreciable numbers of commas, on alkaline agar-agar
jelly. On the following day both cultivations were carefully examined with the
confident expectation of finding colonies of commas present on the one plate
and absent from the other. In place of this, in spite of the most careful and
extended examinations of colonies, no commas could be detected in either
cultivation. There could be no doubt as to the presence of curved schlzomy-
Medical Ojficers of the Army of India.
cete organisms in the original, supposed choleraic material, but there was cer-
tainly a failure in the development of any in the cultivation derived from it. At
this stage another supply of material from the same case came over from the
hospital. As a certain amount of suspicion had been aroused in reg;ird to the
nature of the case, the reaction of this material was tested and found to be acid.
As, however, there was nothing else specially to distinguish it from other mate-
rials, which had yielded commas on cultivation, a fresh plate was set. On the
following morning this was very carefully examined, specimens being taken from
every form of colony which appeared in any way to present distinctive features,
but in none could any commas by detected. The bed-head ticket containing
the history and treatment of the patient was now sent for, and from it it was
ascertained that, although admitted and treated for some time as a choleraic
one, the case had subsequenlly been recognised as one of malarial fever. The
phenomena in this case naturally led to a careful revision of all the others in
which plate cultivations had failed to produce recognisable crops of commas,
and, as has been already shown, in all save one it appeared that there were
either reasons for suspecting the truly choleraic character of the material, or
that conditions were present in the substratum to which the phenomenon might
reasonably be ascribed.
One other case deserves some special notice, namely, one in which, whilst
the original material appeared to be devoid of recognisable commas, a plate
cultivation of it yielded a crop of them. The apparent absence of commas from
the original material may have been due to their being present in v'ery small
numbers and to their having failed to stain w«!l due to the nature of the material
in which they were situated. That the latter condition may exert considerable
influence in certain Instances appears not to be improbable, for I have more than
.once observed that in cases like the one at present under consideration, where the
flocculi were of a very gelatinoid mucous character, a special difficulty in obtain-
ing satisfactorily stained preparations was encountered, the material seeming to
obstruct the stains in penetrating the substance of bodies embedded in it. On
the other hand, however, 1 believe that the failure to detect commas in the original
material may have been really due to the fact that no such bodies were present
in it, for it by no means necessarily follows that because a material contains bodies
capable of giving rise to commas under favourable circumstances, it should
contain actual commas as well. This point, which has already been alluded to,
will be more fully considered subsequently.
Taking the entire series of observations on choleraic materials, the results
certainly appear to indicate that the belief in the presence of easily cultivable
commas as a very constant and characteristic feature in the dejections and intes-
tinal contents in cases of the disease is a well-founded one. They also indi-
cate the absolute necessity of cultivations in suitable substrata in order to the
attainment of any definite conclusions in regard to the presence of bodies of
such nature in any material, for they show In the first place that materials may
8
Scieniijic Memoirs by
be either apparently or really devoid of actual commas and may at the same
time be capable of serving as the source of cultivations containing them, and on
the other hand that they may contain actual commas and yet fail to reproduce
such bodies on cultivation under circumstances favourable to the development
of those associated with choleraic media.
II. — On the presence of Curved Schizomycete organisms
in non-choleraic materials.
A.— On the occurrence of curved schizomycete organisms in normal alvine
evacuations.
An extended series of observations was carried out in regard to the ques-
tion of the presence and characters of commas in such materials.
These included — i, Examinations of fresh materials ; 2, Cultivations.
/. ResitUs of examinations of fresh, normal ahine materials.
Thirty-three distinct samples were subjected to examination, the specimens
being prepared in the same fashion as the choleraic ones. In all of these
without excKption curved schizomycete organisms were present in varying
numbers. The principal difficulty in regard to these materials lies in the enor-
mous multitudes of multifarious schizomycete organisms present in them, which
seem in truth to constitute the greater part of their mass and tend to crowd the
preparations in bewildering profusion.
It therefore becomes necessary to dilute them in order to obtain satisfac-
tory preparations or materials suited for cultivation. This dilution in the pre-
sent instance was effected by means of freshly sterilised salt solution of o'S per
cent, strength.
The number of commas present varied very considerably in individual in-
stances, and in any case was very inconsiderable in relation to that of other
schizomycete forms. They also showed very considerable variation in form.
Many of them were long, slender, only slightly curved and with acutely pointed
extremities. These resembled very closely the commas present in the case
previously described as having ultimately proved to be of non-choleraic nature.
Others, however, presented characters seemingly identical with those of the
commas in typical choleraic media.
In some cases, minute vibrionic or spirillar forms were present in very con-
siderable numbers, and in others large active and energetic specimens of
Amceba coli and Trichomonads abounded.
3. — Results of cultivations of normal aUnne materials containing commas.
In 18 cases primary plate cultivations were instituted. The substratum
in the most of these was neutral or faintly alkaline, but in a certain number of
Medical Officers of (he Army of India.
varj'ing degrees of acidity. Colonies of variotis kinds were developed in abun-
dance and in many cases presenting macroscopic features identical with those
of commatous origin. Specimens were prepared from all colonies which appeared
in any way to present distinctive features. The numbers of preparations from
individual plates in consequence amounted in some cases to as many as ten, but
in spite of this in not a single instance were commas of any kind to be detected.
The forms most commonly developed in the cultivations consisted of short
straight rods and micrococci, but in certain instances an abundant development
of elongated bacilli occurred, the colonies of which were of superficial habit with
peculiar lichenous, foliated margins. The variety of forms developed was
curiously limited in relation to the multifarious nature of those present in the
original materials. Cultivations were not, however, limited to primary plates, for
having become more and more convinced of the polymorphic nature of the
choleraic commas, and of the apparent relation which the appearance of special
forms bears to variations in the conditions in which cultivations are carried out —
the habit of growth acquired in one medium appearing to persist to a certain
extent for some time in others of different constitution — repeated cultivations of
materials derived from the primary plates were made on other plates and in
tubes. The number of successive cultivations made in this way in individual
instances was very considerable, in one case as many as seventeen successive
ones being carried out, but, in spite of continued subjection to conditions
favourable to the development of the commas associated with choleraic media, in
no instance did such development occur here. The nature of the substratum
and the course of cultivation were varied in different ways, the media being in
some cases solid, in others liquid, the reaction being varied, the primary cultiva-
tions being sometimes carried out in liquids, the original materials for inoculation
being sometimes employed whilst fresh, at others only after having been kept
for varying periods, but in all cases the result was alike, there was no develop-
ment of commas.
These results would appear to indicate either that bodies of the nature of
the commas present in choleraic materials are entirely absent from normal
alvine evacuations whether in actual or potential condition, or that, if they be
present, that they are incapable of further development under conditions
favourable to those of choleraic origin. They also point out the absolute neces-
sity of cultivation experiments, showing, as they do, that the mere presence of
curved schizomycetes can neither determine their true nature, nor the character
of the medium in which they exist.
B. -On the occurrence of curved schizomycetes in the intestinal canal of
Guinea-pigs.
Only one or two observations regarding this point were carried out. As has
been pointed out by Dr. Klein, curved schizomycete organisms form a character-
Scientific Memoirs hy
istic feature in the contents of the cscura of healthy guinea-pigs. They appear
to be noimally present in very considerable numbers. Only one series of
cultivations from materials of this nature was attempted and in it no develop-
ment of commas resulted.
C,— On the occurrence of curved schizomycete oreantsnis in water.
The occurrence of commas in water has been frequently pointed out by
other observers, and what [ have lo do here is merely to indicate the connection
existing between certain conditions in the water of tanks in and around Cal-
cutta with the presence of very large numbers of commas which resemble those
associated with choleraic materials in size and form, and so far at all events
in nature in being cultivable in agar-agar jelly.
At almost any season many of the tanks in and around Calcutta are more
or less covered by a scum of Eifglense, which is of a bright brick-red color in
the morning, of "a vivid green in the evening, and which is much less conspicuous
and defined during the day than it is from sunset to sunrise. These variations
in its characters are dependent on recurrent periodic changes in the condition
of the component Esl^lena:. The definition and specially the dry dusty aspect
of the scum in the evening and eaHy morning are due to the fact that at these
times the vast majority of the Englens are aerial and not aquatic organisms,
the cells containing the then encysted and passive protoplasts being raised in
various degrees above the surface of the water, and in the majority of cases being
entirely removed from contact with it and projecting freely into the air. The
relative inconspicuousness of it during the day is, on the other hand, due to the
fact that they are then submerged and swimming free in the water. The
changes in color are dependent on alterations in the relative amounts of red oily
coloring matter, and specially in alterations in its distribution within the bodies
of the protoplasts. The scum is not, however, solely composed of living Eflgle-
nje, but, on the contrary, contains masses of the empty cysts and stems with
dilated bases belonging to previous cycles of the encysted condition of the
organisms. Bright, dry weather tends to induce constantly increasing thickness
in this scum due to the fact that, under such circumstances, the normal cycle of
developmental changes of form goes on recurring with unbroken regularity, and
that accordingly, quite apart from any coincident processes of multiplication
connected with the encysted condition of the organisms, there is necessarily
a constantly recurrent addition of increments of dead matter in the form of
empty cysts and stems. Heavy downfalls of rain, on the other hand, tend to
cause it to disappear, due to the fact that they both break up the sheets of
empty cysts and stems, and by driving the ERglense down into the water tend
to prevent their normal assumption of an aerial habit. So long, however, as
conditions remain favourable lo the regular periodic succession of the diurnal and
nocturnal phases in the life history of the Englenae a steady increase in the scum
Medical Oncers of the Army of India.
goes on. Any scum of this nature, composed in considerable proportion of dead
organic materials affords a favourable site for the development of both saprophy-
tic and parasitic organisms, and we accordingly find it crowded with infusorial,
monadinic, and schizomycete forms. Among the latter curved forms are fre-
quently, and, under certain conditions, apparently normally present in very
large numbers.
In order to obtain these commas with certainty and in large quantity, it is
only necessary to procure a considerable mass of the scum, which can always
be done readily at almost any time from the middle of the cold weather until
the onset of the rains, and to introduce it into a jar of water. The conditions
are here unfavourable to the continued life of the Eflglenas, and they conse-
quently gradually die off, while a very thick scum consisting of their bodies in
various stages of decomposition mingled with masses of their empty cysts and
stems accumulates on the surface, so as sometimes to attain a thickness of
about a quarter of an inch.
If portions of this be removed and their under-surfaces examined, they will
be found to be more or less coated by a stratum of clear gelatinous material
which is crowded with curved schizomycetes of various forms, but many of
which precisely resemble those associated with choleraic media in their morpho-
logical characters.
In two instances in which cultivations of this material were carried out In
feebly alkaline agar-agar jelly, a development of distinct commas occurred. The
subject was, however, unfortunately not pursued further at that time (April) due
to press of other work, and when again recurred to during the rains had to be
abandoned due to the impossibility of obtaining good supplies of the material at
that time. With the return of the cold weather it will, however, be again pos-
sible to take it up and as common gelatine media will then be available for
cultivations, the physiological characters of the commas may be more accurately
compared with those of the choleraic ones than would have been possible at
times when agar-agar jelly has to be substituted for them. In the meantime
the results, so far as they go, must be regarded as enforcing the necessity of
caution in coming to hasty conclusions in regard to the interpretation to be put
on the phenomenon of the presence of cultivable commas in sourcesof water supply.
III.— On variations in the form and course of development of the
Curved Schizomycetes associated with choleraic materials in
connection with variations in the conditions to which they
are exposed.
In taking up this part of my subject ! am well aware that the conclusions at
which 1 have arrived, whilst in accordance with the views of many botanical workers,
are in direct conflict with those of the majority of pathological bacteriologists,
and that I am therefore laying myself open to much adverse criticism in publishing
Scientific Memoirs by
them. This, however, is no sufficient ground for omitting to notice the results
which a series of careful observations have appeared to lead to.
Attention was primarily directed lo this subject first by the observation
that certain modifications in form appeared to characterise many of the
commas which had been developed in substrata which had not been fully neu-
tralised, and which therefore retained a slightly acid reaction, and secondly by
the occurrence in a series of alkaline tube cultivations of a gradual development
of a series of forms similar to those described and figured by Dr. Klein as charac-
teristic of agar-agar cultivations conducted at the ordinary room-temperature in
winter in London.
In the case of several plate cultivations on faintly add agar-agar jelly it
was noticed that, whilst commas were abundantly developed, many of them when
compared with the typical slender commas of neutral or alkaline cultivations
were characterised by their relative thickness and shortness. On examining
them more closely, too, such commas were in many instances observed to pre-
sent appearances which were for some time regarded as indicative of the incipient
stages of spore formation. These consisted in the presence of colorless areas in
the substance of the stained protoplasm (Plate III, Fig. lA). The number of such
spaces varied in individual Instances, being in great part apparently related lo the
size of the commas in which they originated, short specimens containing only one
colorless area whilst longer ones contained two or even three. In general appear,
ance they very closely resembled spores in an early stage of development, but, in
spite of the most careful observation, no further evidences of spore-formation
ever occurred, and it ultimately became evident that they were not due to any
process of that nature but to one of mere vacuolation of the protoplasm.
Numerous observations were now undertaken with a view to determine more
accurately how far variations in the character of the substrata appeared to be
capable of influencing the form of commas developed in them. In the course of
these observations two distinct series of experiments were tried. In the first of
these, cultivations — both on plates and in tubes — were simultaneously started
from a common source, consisting of a pure tube cultivation of commas, in me-
dia of various reactions and the characters of the resulting growths carefully
compared with one another. In the second series the effect of repeated altera-
tions of various reactions was studied, materials derived from alkaline or neutral
substrata being cultivated in acid ones, and the resultant crops being used as a
source of materials for fresh alkaline cultivations.
The result of the first series of experiments was to show that while in neu-
tral or feebly alkaline media crops of typical slender commas were developed,
in the case of acid cultivations these were more or less replaced by shorter and
relatively thicker forms, which in many instances ceased to shew any morpho-
logical evidence of their commatous origin. On comparing the results of parallel
cultivations in feebly and strongly acid media corresponding differences in degree
of deviation from the standard alkaline type regularly manifested themselves.
Medical Officers of the Anny of India.
In feebly acid cultivations a certain, and sometimes a considerable, proportion of
typical commas was present associated with others of shorter, relatively thicker
form and in many instances containing vacuoles. In the larger specimens of
these abnormal commas, the characteristic curvature was still distinctly recog-
nisable, but these were connected by a series of insensibly gradated forms with
bodies which no longer showed any traces of curvature and which presented the
character of rounded or oval cocci with or without a distinct central vacuole-
Such bodies occurred either isolated or in pairs, or in certain instances in con-
nected scries of various numbers so as to give rise to rod-like bodies. The
second series of experiments yielded corresponding results. The transfer of
materials derived from pure cultivations of commas in alkaline or neutral media
to acid substrata resulted in the development of bodies modified as described
above in varying degree according to the character of the substrata as feebly or
strongly acid. Successive cultivations in strongly acid media resulted in the
development of crops almost entirely, or entirely consisting of the modified coc-
cal forms, and successive cultivations of materials of this nature in alkaline media
led to a gradual replacement of the coccal forms by commas and the ultimate
appearance of pure cultivations of typical commas. These results were repeated
with methodical and extreme regularhy as to leave no reasonable ground to doubt
that they really indicated a considerable range of polymorphism as a character
of the choleraic commas, but the precise developmental relations of the various
forms to one another remained for some time a matter of obscurity.
Al length, however, a set of cultivations presented itself which afforded
means for clearing up this question. This, as has already been mentioned, was
one in which the substratum was alkaline and in which peculiar abnormal forms,
similar to those described by Dr. Klein, formed a very -conspicuous feature.
Dr. Klein's observations had cleariy shown that the development of vacuolate
forms was a phenomenon appearing under the influence of certain conditions in
alkaline cultivations. According to his experience the condition which appeared
most closely related to it was depression of temperature. In those cases in
which it was observed in Calcutta It did not appear to be so much primarily
related to thermal conditions, for it occurred in some cases in cultivations con-
ducted during the height of the hot season, as to prolonged cultivation in one
medium. As a rule vacuolate forms were either absent or only present in very
limited numbers in specimens derived from recent neutral or alkaline cultivations,
and where they appeared conspicuously did so in cultivations of some duration
and in constantly increasing numbers as time advanced.
This was very distinctly the case in one cultivation. What rendered this
cultivation particularly instructive was, however, not so much the numbers in which
the vacuolated forms occurred as the exceptionally large size to which many
of them attained, which rendered it possible with comparative ease to follow out
the various modifications of form and to recognise the developmental relations
which the various forms bore to one another. Both the distinctly vacuolate
Scientific Memoirs by
forms and the typical commas present in the cultivation showed very great
range of variation as to size, some being very minute, some of relatively colossal
proportions, and an insensibly gradated series of intermediate forms ser\ing to
connect those presenting extremes of magnitude with one another. The only
difference appreciable between the largest and smallest specimens of the various
forms was one of size. The vacuolate bodies varied very greatly in form, in
some cases being circular, in others almost serai-circular, in others elliptical or
fusiform, and in slill others curved. Some of the curved forms clearly presented
the characters of commas including one, two, or three vacuoles. Others were
thickened in varying degree by filling up of the curve, and where this had
advanced far they necessarily more or less presented semi-circular, hemi-
elliptical, or semi-fusiform contours according to the character of the curve o/
their convex contour. All varieties of vacuolate forms were accompanied by
corresponding non-vacuolate bodies in varying numbers. The circular, fusiform
and elliptical specimens occurred either singly or associated in pairs. The
degree of vacuolation differed greatly in individual specimens, the vacuolar area
in some cases being relatively very large, in others extremely small. In the
former case the precise distribution of the colored protoplasm varied in different
instances. In the case of the circular, and fusiform bodies the vacuole was
centrally situated and the protoplasm formed a more or less even marginal band.
In the elliptical ones the arrangement was sometimes of the same nature, at
others the protoplasm appeared to be accumulated at the opposite poles, being
resolved into two seemingly distinct portions separated from one another by the
vacuolar space. In the case of the commatous, and the serai-circular or other
plano-convex forms the vacuole was either centrally situated or in immediate
relation to the concave or plane edge. In those commas with a central vacuole
the protoplasm was in some cases resolved into two distinct polar bodies, while
in others it formed a continuous rim (Plate III, Fig. ]B).
All the vacuolate forms above described were accompanied by a corre-
sponding series of other forms showing various stages in the process of their re-
solution into commas of various sizes. In the case of the circular bodies of this
class in most instances the peripheral rim of protoplasm could be seen in differ-
ent stages of resolution into a pair of commas, due to the occurrence of a solu-
tion of continuity at two distinct points. The resulting commas were in some
cases of approximately equal sizes, in others one was larger than the other due
to the solution of continuity not having taken place at corresponding points in
the protoplasmic ring. Specimens were present showing varying degrees in the
process of dissociation of the pairs of commas. In many cases they remained
for sometime adherent to one another by one extremity and where they had
done so and at the same time extreme divergence of the opposite free extremi-
ties had occurred figure of three or even V shaped bodies were present. In
other cases, especially in those where the process of resolution tended towards
the development of commas of unequal size, convergence in place of divergence
Medical Officers of the Army of India.
'5
of the free extremities to a greater or less degree had led to the formation of
bodies of concentrically spiral type (Plate III, Fig. i).
Corresponding series of forms showing the process of resolution in ellipses
and spindles were also present in abundance. As in the case of the circles the
commas resulting from the process were either of equal or unequal sizes.
Where the latter was the case the smaller comma often appeared as though in-
cluded more or less within the concavity of the larger one (Plate III, Fig. i).
Very remarkable and instructive appearances were presented in many in-
stances where the process of resolution had affected one of the compound bodies
consisting of two spindles united in pairs by their opposed extremities. Where
the process of resolution had advanced in like degree in both spindles, and
where divergence of the distal extremities of the pairs of resultant commas had
occurred, the entire body presented the appearances represented in the accom-
panying figure (Plate IIlj Fig. iB).
Where, on the other hand, resolution had advanced more rapidly in one
or other spindle, and where, therefore, divergence had only begun to occur in one
of the resultant pairs of commas, this figure was replaced by that also shown in
the figure (Plate III, Fig. iB).
Specimens were also present which appeared to show that in those cases
in which the vacuolation had led to a resolution of the protoplasm into two dis-
tinct polar masses, each of these tended to become gradually converted into a
short comma due to gradual hollowing out of its equatorial face. In this form
of resolution the long axes of the resultant commas will of course be directed
at right angles to the long axis of the parent body in place of parallel to it as
in other varieties of the process. Where vacuolation was lateral in place of
Central only one comma appeared to arise as the result of the occurrence of re-
solution, arising in consequence of gradual conversion of the continuous lateral
band of protoplasm and solution of the vacuolate side of the parent body, the
process being essentially one of rejuvescence and not of multiplicative subdivi-
sion of protoplasm.
The phenomena presented by the various curved forms were parallel to
those occurring in connection with the elliptical and fusiform ones. In some
cases two commas were developed, in others only a single one corresponding
with the convex surface of the parent. Where two commas resulted from the
resolution of a parent body of like form, so long as they retained a definite re-
lation to one another they formed a pair differing from those due to division of
spindles, ellipses, or circles in having the convex and concave faces of both the
commas directed to tin; same side in place of being opposed to one another
(Plate 111, Fig. i). Where processes of resolution affected plano-convex forms
they appeared usually to lead to the formation of a long strongly curved body
and a short straight one corresponding respectively to the convex and plane faces
of the parent. In many cases, however, division appeared to be replaced by
irt Scientific Mevwirs by
rc^jiivcMC'iu !• aiul \\\c. development of a single, large comma only, corresponding
wilii liu» convex surface of the parent.
All these points could be very readily made out in connection with the larger
spcrinuMis of the various forms, and when once this had been done the fact that
siinil.ir proeessc^s took place in the case of the smaller ones also, appeared evi-
di*nt on (MH^ful (examination under sufficiently high magnifying powers.
The* material containing all these various forms was next employed to ini-
tiate a series of lube cultivations, in substrata of various reactions with the
lollowinj; results : —
CtiltiviUioti /. — Siihstratum alkaline. — The resultant crop consisted pri-
inavily in ^;reater part of typical slender commas, many of them being of verj"
minute si/t*. A certain number of small round and elliptical cocci were also
present anil in many of these vacuolation and appearances corresponding with
various stajjes in the process of resolution into pairs of commas were clearly
evident.
(^ultivtiiion II, — From cultivation I ; substratum strongly acid. — The growth
in this ease was mainly composed of cocci. These were either isolated, or asso-
iMated in pairs, or in scTies so as to form compound bodies of rod-like form. Very
few typical eonunas were prc*sent, but many of the longer cocci showed more or
less evident eonea\o-convex contours. Many of the cocci and some of the dis-
tinct cinumas were vacut>late.
(V#//;Vii /;«»>/ llL-'-From cultivation II ; substratum strongly acid — ^The
j;[iowll^ belt* iesenU»led in its characters thai of the previous cultivation, but the
piopouion ol distinct commas was even smaller. Vacuolation was prevalent.
< \>,7;, •.;/;«'// //'. ^'/ew cultivation III ; substratum alkaline. — In this cocci
ol \aiuniv tonns abounded, n^anv of them shewinij various stages of vacuolation
and icM^lutuMi. \'eiv minute commas in many cases associated in pairs were also
piesenl \\\ \A\ys n\MnluM^i.
c \.rV.'..:;';/M l\ /Vi»"; i;<.V/;'.;/.vn* //"; s:*htri:f:,n: alkaline. The growth in
Its eeiu^al ehaiacleis reseiubleil that of the previous cultivation. Some com-
mas ol tan si:e \\eu\ however, preseni amoui^ the minute ones.
(';•/;.. \;;,."; I.'. -/•>»"'; 4V.*V;;'.^';>'; /'.■ s:.rs:*\:t.\r: alialiru\ — The results
beie we\e \e\v msnneti\x\ cotnmas ol vativvas si::es were present in enormous
n\nnbe\s nnn^'jed wnh a eeiiain number ot vwcal forms in various Stages of re-
M«lu((on mio pauNol eontmas.
i '..'.;,;. ',". I.'.'. /"'/": ,':./;;;*;.':/•; •" .', .^;*.\n ;•.;;:.•'; ^rr.^v^'^/v nr/c/.— The
» baiai lets \A \\w i'\o\\\\\ lu\e Wx^ie s'.:n'.;.;: to ihoSi* :v. cu':iva:ion 11.
{\, ,.;;," I . .'.' J.x.* ■•,'••; .....:.;;;,"; ».': .:.\\;/:Vi" j*:.; c^v much diluted
.'\ i\' •. ^v.: ••• ;\ • 'A.;* . ' ;,» ' . ,;' I'bv* i^vew'.h va :h:s oo'^sist^'c of a splendid crop
\-\ KM\e. wbuxMUx tdanu'Mis vxMi^px^-^xx! x^; K*x^'.*.v.v.AS .v.ihc:::":^ :o one another by
lb. u xA\u i\\»nx"» nnox'jxxl \\ul\ a x e:'.ate. e.;:vbo: o: :>,^',i:ixi c.^rr.n:j$. Various
^vtb*'» s»\u*'» x»l xuln\i\uM\'x ei\e \x'N;i!;^ >v:v :'av :.^ '."."^i" dc:,i:.ei above, and
xav.l\il auxl ixpxMUxl xAanu\>ai:.^'aN .^l pwv^ ;•,;.. o ".s .;v:-,\;\: :r::r. :r.e3i have only
Medical Officers of the Army of India.
•7
served to confirm the belief in polymorphism as a character of the choleraic com-
mas under the influence of varying conditions. There can be no question
regarding the correctness of Dr. Klein's observation relative to the occurrence of
processes of multiplication by division of a type differing from the ordinarily re-
cognised transverse one. In some cases the processes are of distinctly longi-
tudinal type and the long axes of the resulting commas then follow the same
direction as that of the parent body, where this is of elongated form. I am
strongly inclined to believe that processes of this character are not peculiar to
the choleraic commas, but will be found to occur in the case of other schiro-
mycete organisms. In a preparation of large oval cocci, developed in a cultiva-
tion of materials from a diphtheritic patch on the surface of a wound in a case of
erysipelas, which is now in my possession, there are, I believe, clear evidences of
the occurrence of similar processes of longitudinal division.
In other cases the process of division is primarily transverse, but at the
same time differs from the commonly recognised process in details and results.
In them we have transverse division of the protoplasm as the result of extensive
vacuolation, and the development of the resultant commas is such that their
long axes are transverse to that of the parent.
These processes of multiplication of commas are initiated by processes of
vacuolation leading to gradual sub-division of the originally continuous protoplasm,
and such vacuolation only occurs under certain circumstances. It tends to
appear, according to Dr, Klein's observations, in association with depression of
temperature ; and, so far as I have been able to ascertain, it is also associated
with partial exhaustion or acidity of the substratum. In recently started alkaline
or neutral cultivations conducted at a temperature of a certain degree of eleva-
tion, such processes of vacuolation and division do not occur, and continuous
processes of transverse division of the ordinarily recognised type lead to the
accumulation of typical commas. Where processes of vacuolative division have
been established, they may be caused to disappear by cultivation under the
conditions favouring the ordinary processes of multiplication. This result, how-
ever, does not necessarily occur completely at once. A varying number of suc-
cessive cultivations may have to be carried out ere these abnormal forms and
processes are completely replaced by the typical ones. It would appear that
we have here indications of the establishment of a special habit of growth in
connection with certain conditions and tending to be propagated hereditarily for
some time under other circumstances.
On the other hand if successive generations of the organism are continu-
ously exposed to the influence of the conditions favouring the abnormal process-
es an opposite result ensues. There is a progressive tendency to disappearance
of the typical forms and to the substitution of abnormal ones differing more and
more from the original type. Taking a material consisting of typical commas as
the primary source of a series of successive cultivations, we obtain various results
3
i8
Scientific Metmirs by
according to the nature of the substrata employed. If the initial cullivation be
conducted in an acid medium, we obtain a growth in which typical commas are
to a great extent replaced by vacuolate commas and cocci of various forms. If
material from such a cultivation be cultivated in alkaline or neutral media the
abnormal forms tend to disappear due to the development of typical commas
due to the ordinary processes of transverse division and to resolution and divi.
sion of the vacuolate forms. On the other hand if the substrata be acid, tho
commatous forms tome more and more to be replaced by vacuolate and coccal
ones. This is due to the fact thai under such conditions, whilst processes of
vacuotation and of division of protoplasm continue to occur, the secondary
protoplasts have less and less tendency to assume the characters of commas and
more to repeat those of the parent cocci.
Vacuolate cocci or short commas, in which the process has advanced so
far as to lead to division of the protoplasm into two distinct polar bodies, will, if
placed under certain circumstances, give origin to pairs of commas, and if placed
under other conditions to pairs of cocci. Where the conditions are such as to
favour growth of a coccal type, the resulting cocci may either separate from
one another or may remain united in pairs or linear series. When they remain
united a series of forms arises which constitute a parallel to the vibrionic forms
which appear under certain conditions in growths of the commatous type.
Such are the conclusions at which I have arrived from observations and ex-
periments of the nature described above. Should they prove to be well grounded,
they will only afford another proof of the absolute necessity of cultivations
carried on under distinctly defined conditions in order to any satisfactory deter-
mination of the question of the presence or absence of schizomycetes of parti-
cular nature in any suspected material.
A few special points still remain to be considered. The first of these
relates to the macroscopic features presented by cultivations of the choleraic
commas in agar-agar jelly. These are not at all of the speciBc and distinctive
type described as characteristic of cultivations carried out in common gelatine
media. There is in no case, so far as I have observed, any tendency to lique-
faction of the basis in the neighbourhood of the colonies. In plate cultivations
the latter are usually evenly rounded, sharply defined and of considerable size ;
sometimes they present a slightly lichenoid appearance. In many cases they
attain a diameter of several millimeters. In one case such defined, colonies
were replaced by a general diffused haze pervading large areas of the plate.
In many cases the colonies are absolutely indistinguishable macroscopically
from those belonging to other forms of schizomycetes with which they are asso-
ciated. Like many other colonies composed of rod-like units they present a
peculiar bluish tint when the plate is held obliquely so as to subject them to a
certain amount of transmitted light, whilst the majority of colonies of coccal
origin under similar circumstances usually show a pinkish, yellowish, or whitish
color.
Medical Officers of the Army of India.
The macroscopic characters of tube cultivations also are neither specific
nor constant. In some cases the track of the needle is sharply defined and
opaque and surrounded either by a series of translucent prominences, which in
profile view form a wavy frill, or by a continuous translucent cloud with well-
defined margins, and acutely conical or clavate inferiorly. In other cases the
needle track presents a number of separate, very minute opaque points and is
acutely conical, wide above and tapering off inferiorly to a point. A translucent
cloud, like that just described, is sometimes present in .these cases also, in
acid cultivations it was usually absent and the needle-track being of the taper-
ing character just described, the entire growth, including the surface halo
around the orifice, presented a tack-like figure. Some of the varieties of the
growth, especially that with defined track and frilled edges, were absolutely in-
distinguishable from those of certain other kinds of schizomycete organisms.
The entire series of cultivations demonstrated the correctness of the state-
ment that, so far as macroscopic characters go, agar-agar jelly is not a favourable
medium for the manifestation of specific physiological properties of the schizo-
mycete organisms cultivated in it.
The influence of the substratum came out very clearly in connection with
the appearance of filamentous forms consisting of commas united in linear series.
Such forms were only very rarely developed in solid media and then only in very
small numbers. In fluid media, on the other hand, they formed a conspicuous
feature, more especially in dilute agar-agar solutions, in which in some cases
to a great extent, they replaced the common isolated commas. In no instance
did the filaments appear to be of truly spiral type. They were merely sinuous
or undulated in various degrees according to the amount of curvature in the
component commas.
The individual commas were, as a rule, of considerable size. The outlines
of the filaments in some cases were alike on both sides due to the concave sur-
faces of the component commas being directed allemalely to either side (Plate
III, Fig. iC.) In other cases there was an absence of bilateral symmetry and
the convexities and concavities of the commas were throughout directed to one
or other side. Here, of course, one side of the filament presented a series of
rounded, and the other of acute prominences (Plate III, Fig. iC.) These
characters were, however, by no means necessarily constant throughout the
entire length of a filament, the arrangement of the commas on which they
depended frequently varying from place to place. It does not appear quite
clearly which arrangement ought to be regarded as the typical one. Where
the multiplication of commas is of the common transverse character, it is
natural to suppose that any filamentous form origination as the result of con«
tlnued adhesion of the commas to one another will be of unilateral type. The
same will naturally be the case in the case of the short filaments represent-
ing the primary pair of commas which have arisen as the result of processes
of longitudinal division in parent bodies of bi-convex outline. Whei-e, on the
3A
20 Scientific Memoir t by Medical Officers of the Army of India.
i — , -_ — .
other hand short filaments arise as the result of similar processes of longitu*
dinal division and divergence in concavo-convex bodies, they will necessarily
be 6f bilateral type.
But filaments of either type may also very possibly arise secondarily as the
result of processes of torsion at the points of union of adjacent commas, so that
in any case it must remain uncertain how far the characters presented by any
given filament ought to be regarded as of primary developmental value, or as
mere indices to previous exposure to certain external influences.
Microscopic examinations of Intestinal, Splenic, Hepatic, and Renal tissues
were onlv carried out in one or two instances. The number was much too
limited to permit of any definite conclusions being arrived at, but in no case did
there appear to be any evidence of the presence of special schizomycete orga-
nisms in the substance of the tissues.
Calcutta,
2Sth September 188^.
On the presence of peculiar parasitic organisms in the tissue of
a specimen of Delhi Boil.
BV
Surgeon-Major D. D. CUNNINGHAM, m.b.,
In the heading given above it will be obser\'ed that the old term "Delhi
Boi!" has been employed in place of the mere comprehensive one "Oriental
Sore." The local designation has been deliberately reverted to, because it
appears to be quite possible that growths presenting the generic characters of
Oriental sore may be due to distinct specific causes in different localities. The
neoplasm in the present instance was certainly associated with, and very pos-
sibly due to, the presence of a peculiar form of parasitic organism. Apart from
this, however, it presented no specific characters, but was essentially a simple
granuloma such as might arise in connection with the presence of persistent
irritation dependent on very various causes. In employing the term " boil "
rather than " sore" I have been guided by the desire to indicate clearly from
the outset that the parasitic bodies in this case occurred independent of the
existence of any raw surface— of any solution in the continuity of the cutaneous
surface — and were present in the initial stage of the disease, in which there is
merely an accumulation of granulomatous neoplasm within the dermal tissue.
There can be no occasion here for considering at length the general condi-
tions in association with which affections of this character arise in India, as this
was fully done a few years ago by Dr. Lewis and myself in a paper on " The
Oriental Sore," since the publication of which no important additions to our
knowledge of the subject have been made. I shall accordingly confine myself
almost entirely to giving an account of the structural features of the specimen
under consideration. This was obtained from Delhi, where it was excised from
the person of a patient in the dispensary, placed at once in absolute alcohol and
sent down to me in Calcutta for examination. In the hard and shrunken con-
dition in which it arrived it measured one inch in length by 0"4i6 of an inch in
maximum thickness. It presented the usual bluntly convex contour, and, as
Scientific Memoirs by
already mentioned, had not yet commenced to ulcerate, so that the term boil
rather than sore was strictly applicable to it.
Vertical sections, including the entire area of the specimen and stained with
picrocarmine, presented the following features when examined under low powers
{vide Fig. i). In the first place it was evident that the portion of tissue which
had been excised did not include quite the whole of the diseased area, for
although at one side the skin presented fairly normal characters, at the other a
continuous invasion of its substance by granulation-tissue was very evident.
The most conspicuous features were the accumulation of great masses of gran-
ulation-tissue, especially in the upper part of the dermis, and excessive hyper-
trophy of the superjacent epidermis. These appearances attained their maxi-
mum development in the most prominent portion of the growth and gradually
tailed of? on either side. On that side, where the limits of the diseased area
appeared to have been approximately reached, the continuous stratum of gran-
ulation-tissue present in the central portions became replaced by isolated patches
of various size separated from one another by normal dermal tissue, and the hy-
pertrophy of the epidermis gradually diminished and disappeared. On the other
side a tendency towards the same thing was evident, but, even at the very ex-
tremity of the section, a thin stratum of continuous granulation-tissue and a
certain degree of epidermal hypertrophy could be readily recognised.
Both horny and mucoid strata of the epidermis showed clear evidences of
varying degrees of hypertrophy. The former in the most prominent portion of
the growth was about o'oi of an inch in thickness, whilst at the relatively nor-
mal extremity it was only about o'ooi. The mucoid stratum was enormously
hy pert rop hied, especially as regarded its interpapJUary portions. Towards the
lateral portions of the sections there was very considerable thickening of it over
the summits of the papilla. But centrally this tended to diminish greatly and
in certain sites an absolute atrophy seemed more or less to replace it, so that the
papillary tissue came to be almost or actually in contact with the under-surface
of the thickened homy layer, and it appeared evident that by a very slight
extension of the process a raw surface would have necessarily been established.
The thickness of the suprapapillary stratum at the extreme margin of the speci-
men, where it presented the most normal characters, amounted to about o'ooi
of an inch, while in the sites of its greatest development it was about 0006.
The interpapillary processes varied extremely in size and form. At the relative-
ly normal margin of the growth, the thickness from the extremity of the process-
es to the surface of the horny stratum was about 0*009 °^ ^" '"'^'^ ; '" ^^*^ most
prominent portions it in some cases amounted to about 007 of an inch, fn
some cases the prominences were broad, in others narrow; in some pointed, in
others distinctly bulbous ; in some they were simple, in others Irregularly
branched interiorly.
Immediately beneath the epidermal stratum there was a great mass of
Medical Officers of the Army of India.
23
granulation-tissue which projected upwards into the papillas and formed a conti*
nuous layer beneath them. Towards the lateral portions of the specimen this
tissue gradually diminished in amount, disappearing from the papilla, and
occurring beneath them in isolated patches only. In the more advanced central
portion of the tumour, the neoplasm appeared entirely to replace the normal
dermal tissue in the area which it had invaded.
This continuous stratum of neoplasm beneath the papillary layer in its
thickest portions was about 006 of an inch deep, and from this it gradually thin-
ned out laterally in both directions. Its inferior margin was in some places fairly
even; in others, on the contrary, it presented a series of irregular processes pro-
jecting for varying distances into the subjacent dermal tissue and there becoming
continuous with a system of irregular masses of similar composition scattered
throughout the interspaces of the fibrous bundles. In some cases these pro-
cesses could be readily recognised as specially related to hair-follicles or to the
ducts of sweat-glands.
The complete continuity of the subpapillary granulation-tissue in its more
developed portions was only interrupted by the bundles of muscular tissue
belonging to hair-follicles which had completely disappeared. In less advanced
portions hair-follicles in various stages of degeneration and disintegration were
also recognisable.
Immediately beneath this continuous stratum of neoplasm was one of modi-
fied dermal connective tissue in course of invasion by the growth. Due to the.
presence of abundant masses of unaltered connective tissue in it the sections of It
stained with picrocarmine presented a conspicuous deep, bright red colour, con-
trasting strongly with the pinkish yellow of the granulation-tissue above it. Inter-
posed between the bundles of connective tissue there were everywhere masses
of granulation -tissue of various sizes and the most irregular outlines. In some
cases these were specially related to hair-follicles, sweat-glands, or the adven-
titia of the larger vascular trunks, but in most they were quite irregularly distri-
buted and appeared as it were to form a natural injection of the lymphatic spaces
of the normal tissue. The appearances seemed to indicate that where hair-fol-
licles have become involved in masses of the neoplasm they are comparatively
rapidly disorganised and soon disappear. The same would appear to be the
case with the straight portions of the sweat-tubes, but the convoluted extremit-
ies, on the contrary, in many instances seem to persist unaltered even where
entirely buried in masses of the growth (Plate III., Fig. 3).
The extent to which the fibrous tissue of the dermis was broken up and, as
it were, dissected by the interposition of these masses of the new growth varied
greatly in different parts of the tumour. Towards the centre masses of the
neoplasm were present in very great numbers and tending more and more to
become confluent with one another. Well-stained sections very clearly demon-
strated the various stages in the process of separation of the bundles from one
H
Scieniific Memoirs by
another and their subsequent resolution into their constituent fibres, the
appearances being similar to those present towards either extremity of the con-
tinuous stratum. This process of interposition of masses of granulation-tissue
and the consequent dissociation of the normal dermal elements necessarily gives
rise to very considerable thickening, and it is to it along with the constantly
increasing thickness in the accumulation of continuous granulation-tissue abovet
together with the associated epidermal hypertrophy, that the gradual increase
in prominence of the affected area of the skin is due.
Beneath the layer of modified dermal tissue the sections included fat-lobules
of the subcutaneous areolar tissue, which, like the structures above them, showed
in varying degrees distinct evidences of invasion by granulation tissue. In the
lobules immediately abutting on the dermis masses of the neoplasm in the latter
spread continuously into the substance of the lobules, dissecting the fat-cells
apart from one another, pressing upon them in varying degree, and ultimately
obliterating and replacing them entirely throughout considerable areas. Scat-
tered and, so far as the sections were concerned, isolated patches of various
size were also scattered throughout the lobules in varying numbers. In many
cases these seemed to be related specially to the adventitia of the larger lobu-
lar vessels ; in others, however, they appeared to be scattered at random among
the fat-cells. Even in the very deepest portions of the specimen such Isolated
patches of neoplasm could in many places be recognised in the lobules (Plate
III., Fig. 2).
Examinations of similar sections under higher microscopic powers confirmed
the information attainable by means of low ones and revealed additional points in
regard to structural details. The essential feature in Ihe diseased area was clearly
shown to consist in an accumulation of lymphoid and epithelioid cells among the
normal tissue elements. When Dr. Lewis and I previously studied the subject,
we were inclined to regard the morbid process essential to the disease as one in
which a development of tissue of an adenoid type occurred. Now, however this
may be so in certain cases, it most certainly is not necessarily the case in all,
for in the present one there was no evidence of the development of any new
stroma — of any new fibrous network. The most careful examination of sections
both before and after brushing showed nothing, save an accumulation of
lymphoid and epithelioid elements between the normal connective tissue struc-
tures of the dermis and subcutaneous areolar tissue, together with the results of
associated processes of epithelial hypertrophy. As has been already pointed
out, the morbid process In the dermis consisted essentially in a simple opening
out and dissociation of the normal tissue elements by masses of pure granulation-
tissue. The ultimate result of any such process must naturally be, where it _
advances sufficiently far, to replace the dense connective tissue of the dermis bjs
one consisting of a stroma of separated, but persistent fibres Including deni
masses of cellular elements. The size and form of the spaces in the stroma
Medical Officers of the Army of India.
25
naturally varies greatly in different instances; in some they are relatively very
large, as they must almost inevitably be where corresponding to interfascicular
fareas ; in others they are much smaller and include only small masses of cellular
I elements, which may be either spread out in simple strata so as to form plates,
or be heaped upon one another in irregular masses. Plates consisting of a
single stratum of cells will naturally on vertical section appear as linear series
of cells, whilst the forms presented by the aggregates of other nature will
necessarily vary with the relation which the p!;me of the section bears to their
actual plan. A certain number of fusiform protoplasmic bodies resembling the
nuclei of typical connective tissue corpuscles may no doubt be recognised
throughout the substance of the neoplasm (Plate II., Fig. 5). The number of such
bodies in the.present case was, however, very trifling, and it appeared much more
reasonable to regard them as mere persistent elements of the normal fibrous tissue
than as indices to the occurrence of any processes of development of a neoplastic
stroma. Repeated and careful examinations of very many sections treated with
I numerous distinct staining reagents have only served to convince me more and
more strongly that, if any development of new fibrous elements occurred at all
in connection with the diseased process in this case, it could only have been
very trivial, and that the real stroma of the neoplasm consisted merely of dis-
sociated elements proper to the normal tissue of the part. The characters of
the specimen appear to me clearly to show that a development of adenoid tissue
is not necessarily a feature in the disease ; they do not, however, indicate that
I such a process may not occur in certain, or even in the majority of, cases.
The cellular elements forming the essential feature in the neoplasm vary
Pconsiderably both in size and outline (Plate II., Figs. 9 — 14). This is apparently
to a considerable degree determined by the varying degrees of pressure to which
they have been exposed during and subsequent to their development. In those
sites in which they have not accumulated to any great extent, where they lie more
or less separately in the spaces in which they are situated, many of them are more
or less rounded in outline, or where they present an angular contour, are of con-
siderable size and epithelioid character. Where, however, a greater accumulation
has occurred, and where, therefore, the cells within the meshes of the stroma
have come to be closely packed together, they are almost without exception
of small size and more or less angular from mutual pressure. Where the
inter-spaces in the stroma are of small size and the accumulation of such minute
angular elements is relatively excessive, the outlines of the individual members
of the aggregate may be very hard to distinguish, and the entire mass may thus
come closely to simulate a single, large, polynucleate cell (Plate II., Fig, n).
Such densely aggregated masses of cells, as was previously pointed out by Dr.
Lewis and myself, appear to form one of the sources from which the peculiar
concretionary bodies occurring in the discharges in cases which have passed on
into ulceration are derived. Truly polynucleated elements appeared to be en-
tirely absent, and even binucleate ones were very rare.
4
s6
Scientific Memoirs by
The average size of a series of cells which were carefully measured was
8'8^ X 7*2^, but the variations between individual specimens was very consider-
able, the largest ones measuring as much as io'7iu.x8'5/i, the smallest only
5'4pX5"4/i. The size of the nuclei was much more constant to an average of
5"6/iX 3'4/Li. In any case they were relatively large, and in the case of the
smaller elements the layer of cytoplasm was so thin as to cause them in many
instances to simulate free nuclei.
Examinations under high powers also demonstrated the various stages in
the development of the growth very clearly, showing the gradual accumulation
of lymphoid elements with progressive dissociation of the normal structures of the
invaded area. The appearances presented in many of the fat-lobules of the sub-
cutaneous areolar tissue were particularly instructive (Plate 111., Fig. 4). In some
places only a few scattered lymphoid and epithelioid elements were present be-
tween the fat-cells ; in others distinct local accumulations had occurred ; in others
such accumulations had led to separation and partial compression of the sur-
rounding cells, and in still others continuous masses of lymphoid and epithelioid
elements had entirely replaced the fat-cells throughout considerable areas of the
affected lobules. The adventitia of the lobular vessels, as before mentioned, ap-
peared in many instances to form the primary site of invasion, but in others this
was not the case, the adventitia remaining quite free even where other portions
of the lobule had been seriously affected.
In the areas of invasion of the dermal tissue proper appearances corre-
sponding with a natural injection of irregularly ramified lymphatic spaces were
in many cases most distinctly traceable. In other places the lines of invasion
had clearly primarily followed the tracks of hair-follicles or the ducts of sweat-
glands. The various stages leading to the formation of the epithelial concre-
tions occurring in the discharges of ulcerating specimens of the disease in asso-
ciation with the other form of concretions previously alluded to, could be readily
traced as connected with processes of continuous growth occurring in the tips of
inlerpapillary processes, or of detached portions of hair-follicles which had be-
come isolated and buried In masses of the granulation-tissue (Plate III., Figs. 5,6).
It is unnecessary here, however, to go into further details regarding the
general structural features of the specimen, as these fully agreed with those des-
cribed by Dr. Lewis and myself in our previous paper, and I shall therefore now
go on to the special consideration of the characters of the peculiar parasitic
bodies present in it, and which it appears at all events possible were the primary
cause of the disease.
The specimen was primarily procured with the view of ascertaining whether
there were any evidence of the association of any special form of Bacterial
organisms with the diseased tissues. A very large number of sections were
accordingly prepared with the freezing microtome and were subsequently sub-
jected to the influence of various staining reagents — Picrocarmine, Logwood,
Medical Officers of the Army of India.
»7
Gentian violet, Methyl blue, Spiller's purple, Bismarck brown, Fuchsin, Eosin,
and Gibbes' double-staining fluid.
The specimens thus prepared were subjected to careful and methodical
examination, but in none of them could Sthizomycete organisms of any kind be
detected. In none of them, save those which had been stained with Genlian
violet, did any structural features manifest themselves beyond those which have
been just described above. In those, however, which had been first thoroughly
stained with Gentian violet and then washed out in spirit until almost all colour
had disappeared from them, large numbers of peculiar violet or blue bodies
appeared conspicuously among the surrounding, almost colourless masses of
lymphoid elements (Pi. [., Figs, i, 2; PI. II., Fig. 8). Repeated and careful
examination of a large series of sections prepared in a similar fashion revealed
the constant presence of such bodies and furnished the data for the following
details regarding their structural features and distribution.
In preparations stained with any of the other dyes mentioned above, with
the exception of Fuchsin, they were absolutely indistinguishable even after a
complete familiarity with their appearance and distribution had been attained.
In some Fuchsin preparations faint indications of their presence could be recog-
nised, but it was only by means of Gentian violet that they were rendered con-
spicuous and their characters sharply defined. Double staining with Gentian
violet and Fuchsin in some cases gave very good results as will be further allud-
ed to presently.
They varied very considerably in size. The average diameters of a series of
measured specimens were \2-6fi. x 8"S^, the largest measuring I2"8^ x 25"6/i,
the smallest 6'4ia x 6"4/i. Such minute specimens as the latter were, how-
ever, rare, and, as a rule, they were considerably larger than the lymphoid
elements among which they were situated. Their form also varied greatly, — in
some cases they were circular, in others elliptical, in others irregularly lobate
(PL i. Figs. 3,3, PI. II. Figs. 3, 5, 6). Their contour was in the majority of ins-
tances smooth, but in some of a more or less tuberculate character. In some
specimens a very delicate cell wall was clearly visible; in others it was wholly
unrecognisable, or only to be detected on careful and special scrutiny.
The distinctness with which they appear in sections treated with Gentian
violet is due to the elective staining of the nucleoid bodies which they contain
by the dye. The number of such bodies present in different cells varies ex-
tremely, — in some cases only a single great nucleoid mass is present, seemingly
occupying almost the entire cell body, in others a few of very various sizes
occur, and in still others a large number of minute and fairly equal-sized ones are
thickly scattered throughout the entire cell (Plate I., Fig. 3 ; Plate II., Figs. 3 — 7).
The cytoplasm in the Gentian violet specimens remains almost uncoloured ; in
those in which Fuchsin has likewise been employed, it frequently shows a more or
less pronounced red hue. The tuberculate appearance presented by some of the
Scieniijtc Memoirs "by
cells is due to the numbers and size of the nucleoid bodies present in them,
which in association form a muIberry-Uke mass pressing upon the cell wall and
moulding it to the inequalities of lis surface (Plate II., Fig. 2). Such lubercu-
late bodies on superficial examination present certain points of resemblance to
the characteristic bodies in cases of Actinomycosis. On closer examination,
however, it is evident that they correspund structurally with the description
given above and are not due to any radiate aggregation of filaments.
In certain cases appearances apparently corresponding with the occurrence
of processes of cell division are present, the bodies of the cells being strongly
constricted so as to form two lobes connected by a narrow neck, or two distinct
cells occurring which, from their relations to one another and the character of
their opposed surfaces, seem to have just arisen due to the completion of such
a process (Plate II., Figs. 4,5). In many instances, too, a distribution of the
cells in little groups separated from one another by comparatively wide areas of
granulation tissue can be recognised, and may possibly be indicative of the
antecedent occurrence of processes of division (Plate I., Fig. i).
The individual cells in some cases are closely packed among the surround-
ing lymphoid elements ; in a large number of instances, however, they appear to
lie in a limited clear space (PI. II., Fig. 4,6). This appearance may possibly be an
artificial one arising as the result of shrinking during the course of preparation.
The number of cells visible in individual sections and in different parts of
the same section varies considerably. In some sections as many as eighty or
even more may be visible at once in a single field under a power of one hundred
and forty diameters. In others they are present in varying but smaller numbers,
and, in almost any, entire fields may in certain places fail to show any at all.
This failure may in many instances be due to imperfect success in staining, but
allowing this, there can be no doubt that the numbers present in different parts
of the tumour vary greatly.
It is only quite exceptionally that any are present in the epidermal stratum.
In one or two cases, however, I have detected specimens in the substance of
interpapillary processes in sites which they could not have reached by acciden-
tal transfer during the course of the preparation of the sections. The conti-
nuous stratum of granulation-tissue beneath the papillary layer is the site in
which they occur in greatest numbers, but specimens are also frequently present
in considerable numbers within the papillary eminences. It is in the central,
thickest portion of the continuous stratum that they occur in greatest numbers,
and in passing outwards laterally from this they tend to appear in progressively
smaller numbers, until towards the edges entire fields apparently devoid of them
may be encountered. In those sites where traces of halr-foIHcles remain re-
cognisable they are often present in specially large numbers (Plate II., Fig. 8).
In the portion of the tumour next beneath this, where the neoplasm is no
longer continuous but is interposed in irregular masses between persisting por-
Medical Officers of the Army of India.
39
tions of normal dermal tissue, numerous specimens are also present. Here also
as In the superior stratum they frequently are specially conspicuous in the
neighbourhood of altered hair-follicles. The large masses of neoplasm around
the sweat-glands and larger vascular tninks, loo, often contain an abundance
of them, while smaller numbers or isolated specimens are present in many of the
masses of neoplasm generally distributed throughout the entire system of
interstitial spaces. Here, as in the upper stratum, it is in the central portions
of the tumour that they occur in largest numbers.
'Iheir distribution is not, however, limited to the epidermal and dermal
strata, for on passing downwards to the subcutaneous tissues scattered specimens
may be. found even in the very deepest parts. The numbers actually present
in different sites varies in proportion to the degree to which these have become
the sites of invasion by masses of lymphoid elements, but even where this has
taken place only to a very limited extent, isolated specimens may be encountered
(Plate I., Figs. 6, 7). For example, the specimens shown in Plate I., Figs. 4, 5 ;
Plate 11., Fig. i.are situated in one of the interstitial spaces between the cells in
fat-lobules in which an accumulation of lymphoid elements has only just begun
to occur.
There can, then, be no doubt that in this specimen of " Delhi Boil" these
peculiar bodies are generally distributed throughout the entire extent of the
morbid tissue, and that the numbers of them present in particular areas corre-
spond generally with the degree to which the morbid process has advanced there.
It remains now to consider the question of the nature of these bodies and
of the relation which they may be regarded as holding to the disease.
Unfortunately it is as yet impossible to come to definite conclusions on
either of these points. The cells have as yet only been observed in preserved
tissues, and without dehnite data regarding their development and life-history
generally the utmost that can be done is to indicate probabilities regarding their
nature. After very careful consideration of the features presented by all the
various forms present in the tissues, I am inclined to regard them as represent-
ing various stages of some simple organism of Mycetozoic nature. In the most
recent systematic treatise dealing with the Mycetozoa or Myxomycetes — ZopFs
" Schleimpilze" they are subdivided into Monadinx and Eumycetozoa, and
it is to the former group that the organism here dealt with appears to me pro-
bably to belong. The appearances presented by the various forms are, accord-
ing to this view, to be regarded as corresponding to various stages of the de-
velopment, and specially of the development of the Zoocysts or Sporocysts,
of some Monadinic organism. Comparing the characters of the various speci-
mens with one another, we have apparently to deal with the development
of parent plasmodia or amoebs which multiply by division, and in which sporoid
bodies are gradually developed, the process terminating in some cases with the
formation of one great spore, in others with that of a dense aggregate of smaller
30
Scientific Memoirs by
ones. As has been just indicated, however, we are here dealing with probabi-
lities only, and any definite conclusions can only be looked for after the organisms
have been studied while alive, and the various stages in their life-history
actually followed out in cultivations in suitable media.
The necessity for such observations is even more urgent in order to the
attainment of any definite answer to the question of the relation which they bear
to the occurrence of the disease with which they were in this case associated.
There are, however, certain points rendering it possible, if not even probable
that they actually were the specific causes of the disease. In the first place,
there can in regard to this specimen be no question of accidental entrance of
the parasites into the morbid tissues due to abnormal facilities presented by the
surface, for there was no ulcerated, raw surface present, and, on the contrary,
the homy layer of the epidermis was abnormally thickened and resistent. This
in itself is in favour of a belief in their causal relation, as it is hard to see why
such bodies should specially attain access to the diseased rather than to the
healthy tissues unless some special structural facilities are provided in the former
for their doing so. The general features of the distribution of the parasites
also points in the same direction, for, as has been pointed above, they are
found to occur in numbers varying in fairly close correspondence with the degree
to which morbid changes have taken place. Much more significant, however,
is the fact of their frequent presence in sites where such changes are only
beginning to manifest themselves — In sites where small, isolated aggregations of
the neoplasm are interpolated among wide areas of normal tissue elements.
The circumstances under which the disease generally tends to occur — its
markedly endemic character, and specially its connection with the employment
of certain sources of water supply — are also strongly in favour of the causative
agency of bodies of the nature which those in the present case apparently
possess, as the plasmodia and zoospores of the Monadinse are specially likely
to be present in water of special localities and of particular characters. The
distribution of specially large numbers of the parasites in special relation to the
remains of hair- follicles and in the neighbourhood of sweat-ducts is also of
some importance as it seems possibly to indicate these structures as special
channels of access to the substance of the tissues, and naturally they can only
serve as such so long as they retain their normal characters and have not been
pressed upon and obliterated by surrounding accumulations of neoplastic ele-
ments. The fact that the parasites occur in such deeply-seated areas as the
subcutaneous fat-lobules is quite in accordance with the clinical experience that
no superficial application of remedies is effectual in putting a stop to the disease,
and that free excision or penetrating destruction of the tissue of the diseased
area is necessary in order to do so.
It may, moreover, be pointed out that there are well-ascertained instances
in which parasites of monadinic nature ser\'e as the efficient cause of the de-
Medical Officers of the Army of India. 31
velopment of tumours in the host body which they affect, not merely as the re-
sult of accumulation of their own proper elements, but due to the occurrence of
coincident processes of excessive hypertrophy in the host-tissues apparently of
irritative origin. For example, in the case of Woronin's Plasmodiophora Bras-
sicae we have to deal with a parasite of this nature affecting various species of
Brassica and causing both great hypertrophy of individual cells and accelerated
rate in processes of cell division in the host.
The data which have been attained as the result of this examination of a
specimen of typical Delhi Boil appear, then, to me to show that the diseased
processes in cases of so-called " Oriental Sore " may certainly be associated
with and possibly caused by the presence of peculiar parasitic bodies. In
regard to this, however, it appears to be quite possible that such bodies may
constitute a real cause of such disease without necessarily constituting its
only cause. The neoplasm in specimens of Delhi Sore, such as the present one,
presents no specific characters. All the changes in the normal tissues may be
reasonably ascribed to mere persistent local irritation of certain intensity, and it
is quite conceivable that such irritation may be connected with different
exciting causes in different instances. Even, therefore, should it eventually be
demonstrated that all cases of such disease occurring in and around Delhi are
invariably and causatively related to the presence of the parasitic organisms
here described, it by no means necessarily follows that all similar forms of disease
occurring in other localities should have an identical causation. It is quite con-
ceivable that similar morbid changes may arise as the result of the presence of
other forms of parasitic organisms, or may even be developed apart from para-
sitic agency of any kind whatever,
Calcutta ;
The 1st October 1885.
«.>|i^'«
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PARASITIC ORGANISMS IN A SPECIMEN OF DBLHI-SORB.
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