NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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some of the flagellated forms found on the fourth day in case 47, in addition to
which there were much more numerous double pyriform organisms without flagella,
for only a very small percentage of the conjugating forms eventually reach
the flagellate stage under the artificial conditions of the cultures, which must be
very far from being as favourable to the development of the organism as the
natural conditions in which it takes place, whatever it may be."
With regard to elongate forms, Captain Rogers notes that, though they
occurred in several of his cases, yet they did not always appear. He also
notes that the extremely long attenuated forms figured by him in his first publica-
tion are exceptional, both in that elongation was more marked than usual, and in
that this great degree of development had taken place in 24 hours.
In the main I have been able to confirm Captain Rogers' results. Indeed,
most of the forms figured by him, including very elongate forms, have occurred
in my preparations. In addition I have been able to observe certain further
appearances not described by him, which appear to take place at a later date,
and to add some details of description. On the other hand, I have been unable
to accept his conclusions regarding conjugation of the large oval forms or to be
convinced that the nature of the bodies is yet finally settled.
Technique .—At first I made several experiments with weak solutions of
citrate of sodium, i.e., from .5 to 2 per cent. without success, but since I was
informed that Captain Rogers used a 10 per cent. solution, I have employed this
strength, and it is with this stronger solution that positive results have been
obtained. I have found the following the most convenient method of observing
the developmental changes.
A 10 per cent. solution of citrate of sodium in distilled water is freshly
prepared. The salt used in my experiments was obtained by very carefully
neutralizing pure sodium hydrate with citric acid. The fluid, after concentra-
tion on a water bath, was again neutralized and crystals allowed to form. The
crystals were redissolved and the salt recovered by a second crystallization.
Some time before the operation of spleen puncture is to be carried out, a
Burroughs and Wellcome "all-glass" syringe (small size) fitted with a platino-
iridium needle is rinsed out with the citrate solution and placed in a test tube with
the piston resting upon a pad of wool. About .5 c.c. of citrate solution is left in
the syringe and the cotton pad is damped with the same. The test-tube is
plugged and the whole sterilized at 120° C. for half an hour in the autoclave.
At the bedside the test-tube is tilted to allow the syringe to fall needle
downwards against the plug. The plug is then removed far enough to allow
the barrel of the syringe to be seized by the fingers. It is convenient to have
three or four prepared syringes at hand, in order to reduce the amount of pre-
paration necessary at the bedside.
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Immediately before puncture any excess of fluid is ejected from the syringe,
only a little being allowed to remain in the needle and nozzle. A successful
puncture should one-third or one-half fill the barrel of the syringe.
Illustrating author's method of studying development
of bodies in citrated blood.
Blood from the syringe is ejected with the fullest precautions as to sterility
into small sterile tubes. I have found "arsenic tubes" (straight 3 "X3/8" ) v e r y
suitable, their narrowness and length being antagonistic to desiccation and
contamination during the frequent examination of the blood. For steriliza--
tion these tubes may be heated to 120° C. in the autoclave, or merely passed
through the flame till "browning" of the cotton plug takes place. No citrate
solution is added to the tubes. After blood has been ejected into the tubes, I
have found it convenient to enclose them in a small tin canister containing a
little damp wool and provided with a tightly fitting lid.
As I was obliged to use an ordinary ice chest, the cultures were exposed to
a somewhat varying temperature, but it did not rise above 24°C.
Blood for examination is removed by means of a platinum loop, great care
being taken each time to prevent bacterial contamination. As the tubes
employed were long and narrow, it was necessary to use a long, rather thick,
platinum wire instead of the ordinary loop with a holder.
Before removal of a drop for examination, the blood is shaken up to mix the
corpuscles and plasma thoroughly. This enables more satisfactory films to be
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made as well as ensures parasites being equally distributed. In good preparations
the blood corpuscles remain unchanged for 15 days or more : they cannot be
distinguished under the microscope from the corpuscles of freshly drawn blood.
The leucocytes show marked degenerative changes, and the nucleus becomes
swollen and irregular. Nevertheless in some cases the variety of cell could be
distinguished easily after several days.
Development. —Development of the bodies was observed in four cases.
In one the preparation was examined every few days for a period of 33 days, at
the end of which time the corpuscles were but little altered and no micro-
organisms were seen in the films. The remaining cases, owing to the pressure
of other duties, were not followed up so systematically, and in two of the cases
commencing changes only, though of a convincing nature, were seen. The
results noted are as follows :
—
Case 1.—The patient had a greatly enlarged spleen reaching across the
middle line of the abdomen and an irregular high temperature reaching to 102°F.
and 103°F. at night. About .75 c.c. of blood was removed from the spleen in
the manner already described. In films made at the time of puncture parasites
were fairly numerous. The specimen was surrounded by ice, so arranged that
the preparation should not be made too cold, and carried about six miles ; it
was then placed in the cool chamber of an ice chest at a temperature varying
between 22°C. and 24°C. No clotting occurred, and when shaken up the blood
retained for weeks a natural appearance.
The first unmistakeable sign of development was seen on the third day.
On this day, although the majority of the bodies appeared unaltered, yet a
considerable number were somewhat larger than usual. A few were also
noticed in which the protoplasm had increased in amount and had stained
a dark blue. The large chromatin mass had also increased in size, and this as
well as the smaller mass, occupied a more central position. The appearance of
these forms was unlike anything I had previously seen in preparations direct
from the tissues.
On the fourth day no doubt remained that a remarkable development was
taking place. From Captain Rogers' description I was not prepared for such a
great increase in size as occurred, nor for such startling changes in the appearance
of the bodies. On this day, although many forms were still unaltered, very
many had undergone the change noted above, whilst others had increased four or
five fold and, though still retaining the characteristic double chromatin masses,
had an appearance totally unlike the original bodies.
Large forms.—The large forms, which had apparently risen each from a
single parasite, were fairly abundant and many examples were readily found.
In most cases they were as large or larger than the red blood corpuscles,
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measuring in some instances as much as 8
i n d i a m e t e r . A s a r u l e , t h e b o d i e s
were slightly oval or bean-shaped. The protoplasm was finely reticular and
stained a distinct blue. The chromatin masses still retained their characteristic
arrangement and appearance, but were situated well within the protoplasm,
instead of being upon the periphery. The large chromatin mass had increased
in size and had become granular. In some cases the granules showed a very
regular arrangement. The small chromatin mass appeared unaltered or slightly
increased in size. It showed most usually the rod-shaped form seen in the original
bodies. These forms are undoubtedly those described by Captain Rogers as
occurring on the third day, though this observer has not laid very great stress
on the extreme enlargement which has taken place in the bodies since their
removal from the spleen. No double forms were seen on this day, but commen-
cing fission was already seen in some, an observation not in favour of the
conjugation view held by Captain Rogers.
Fifth day. —Large forms resembling those just described were readily found.
Bodies with double small and large chromatin masses and commencing fission
forms were not uncommon. Most frequently the bodies occurred in groups of two.
Groups of four were also seen, and in some instances it was clear that these had
arisen from a second fission of groups of two. I could find no evidence that the
double forms represented conjugation ; it seems much more likely that they are
the result of the first fission process.
As these forms lying in groups of two and four have certain characters not
seen in later development, I have termed them, for the purpose of description,
"primary fission forms."
Primary fission forms. —Though most frequently these forms lie close
together in the preparation, they show no trace of the cement substance so charac-
teristic of the later fission forms. In some cases a faintly staining substance
retaining the bodies in approximate apposition can be made out, but it is
entirely distinct from the red staining substance seen later.
Each form is of large size, oval or bean-shaped. A distinct pear shape is
not seen at this stage. Fission occurs at least twice before further development
of the forms to be described occurs.
The formation of elongate forms. —One or two forms were seen on the fifth
day in which fission had resulted in two rather elongate bodies with the small
chromatin masses in opposition to one another and occupying one extremity of
the parasite.
The formation of a vacuole-like area.—In certain of the forms, apparently
after fission had produced at least four separate bodies, a new structure was
visible. This structure is not mentioned by Captain Rogers, though it is faintly
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indicated in one or two of his figures. It is seen as a pink staining area about the
same size as the large chromatin mass. It lies generally in the neighbourhood of
the small chromatin mass. It appears to be of the nature of a vacuole, but con-
tains strands or flocculi of a material which stains light pink.
In all later stages a proportion of the parasites showed this appearance
more or less clearly.
The extrusion of the cement substance.—On the fifth day very curious forms
were seen in which a red staining substance of a peculiar "woolly" appearance
seemed to be poured out from the parasite. This was first noticeable as a tail
-like process projecting from the end of the parasite in which the small chromatin
mass is situated. In some instances the origin of this streak appeared to be in
close connection with the small chromatin mass, and in very early stages it
might be mistaken for the commencing formation of the flagellum. Later the
substance becomes more voluminous and forms are seen in which it appears to
be pouring out into the plasma (fig. 13). The substance stains at first a bright
red, but when in large masses is often of a dark red colour. This substance
increases in volume very much, and eventually surrounds the end of the parasite
in which the small chromatin mass is situated. Afterwards it fuses with that
thrown out by adjoining parasites and binds the members of a group firmly
together (figs. 17 and 18).
The formation of the flagellum.—On the fifth day no distinct flagella were
seen as on the sixth day and later. One or two bodies, however, showed an
appearance which may indicate the method of formation of the flagellum. Apart
from this, I have not been able to trace any stage of the flagellum prior to its
appearance in a complete form. In certain of the bodies seen on the fifth day a
faintly stained, long, but rather ragged, filament extended from the end from
which the flagellum afterwards appears. The colour taken up by this filament
approximates almost exactly to that of the pink vacuole-like area, and in several
instances the protrusion of this delicately staining filament appeared to be
associated with the passage of the vacuole to the surface (fig. 12).
The filament, though clearly distinguishable, was quite unlike the defined
rod-like flagellum which is seen later. It is most probable that the faintly
staining filament represents the flagellum when first extruded.
In later forms, when the cement substance has obscured the view, it is very
difficult to ascertain what is happening at the end of the parasite containing the
small chromatin mass.
Sixth day. —On this day isolated forms were quite exceptional and the
bodies were seen lying in groups of from four to ten, groups of four or five
being most common. The individual forms were still large and conspicuous,
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being rarely less than the size of the red cell. In almost all instances large
masses of red staining material had been thrown out.
Most of the bodies were pear-shaped or elongated. The extremities of the
parasite containing the small chromatin masses invariably lay close together,
whereas the rounded swollen portions projected freely. The pink vacuole was a
conspicuous feature in many of the forms, and others possessed distinct, clearly
outlined, deeply staining flagella.
Flagellate forms.—On the sixth day almost every group contained one or
more flagellate forms. In some cases all the bodies composing the group had
developed flagella. Occasionally isolated flagellate forms were seen, but they
were not common. The attached end of the flagellum could, as a rule, be
traced to the small chromatin mass or to its close neighbourhood. The
small chromatin mass thus appears to be a blepharoplast. In many cases it
was difficult to trace the flagellum to its origin on account of the cement
substance. At the free end of the flagellum a small but distinct knob was very
often visible. The length of the flagellum varied, being from 12 µ to 30µ in
length. The flagella, especially the shorter ones, appeared rather stiff and rod-like,
though in some cases they had an undulating outline. On the sixth, seventh and
eighth days fission produced both large and elongate forms (fig. 16). In some
cases the elongate forms were very striking and trypanosome-like ; but in no
case did the flagellum pass forwards along the large chromatin mass. As a
rule, the greatly elongate bodies formed members of grou ps in which the majority
of forms were much less attenuated. In all cases the small chromatin mass
lay towards the centre of the groups, and flagella when present passed directly
from this end (fig. 16).
Development of bodies included in cells .—On the sixth day and, indeed,
throughout the earlier stages it was not unusual to find bodies undergoing
development whilst included in the altered substance of a cell. As development
progressed, and a group of large forms resulted, these projected so as to give
rise to a fungating like mass of the bodies (figs. 20 and 21). The growth of forms
which are undoubtedly included in a cell is interesting in relation to the fact that
such forms are in the majority in the spleen. It would appear that an included
form is not necessarily a dead one.
On this day an unstained drop of the citrated blood was examined, but
though several groups of hyaline nucleated bodies were seen no trace of flagella
or appearance suggesting motion of these organs could be made out.
Seventh day .—On this day groups composed of many forms were the most
common. Most of the groups contained several flagella-bearing individuals.
The red staining cement substance was very voluminous and conspicuous. Many
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of the forms showed the pink staining vacuoles already described. Elongate forms
were not more numerous than on the previous day. Fission appeared to be
proceeding actively.
Ninth day .—On the ninth day isolated forms were rare and the bodies
occurred almost exclusively in large aggregate masses. Elongate forms were
exceptional and most of the parasites were pear-shaped or polygonal from mutual
pressure. The cement substance was very voluminous, and flagella were some-
times seen entirely embedded in it (fig. 17). Short thick flagella were generally to
be seen in a proportion of the individuals of each group. Fission appeared to
be taking place more irregularly than heretofore, and difficulty was often
experienced in tracing out the boundaries of individual forms and in assigning to
each their respective chromatin masses.
Secondary fission forms. —On the sixth day it was evident that fission of
the bodies was proceeding rapidly, and from this day onwards fission, leading
to the formation of larger and larger masses, composed of bodies of various sizes,
was one of the chief features of development. This final condition of repeated
fission is not noted by Captain Rogers. It appears to follow, in some cases at
least, upon the formation of flagellating pairs, which is the most advanced stage
described by this observer. Considerable difference in appearance exists
between the large oval forms which I have described as primary fission forms
and the bodies now under discussion. From the ninth day onwards fission
tended to produce smaller and smaller as well as more irregular bodies,
which may be conveniently termed secondary fission forms.
Twelfth day. —Sub-division had still further progressed. Individual forms
were often small, though still larger than the original bodies prior to development.
The general appearance of the developed forms was by this time entirely unlike
the parasites as drawn from the spleen, and had not all intermediate forms been
seen, their identity would scarcely be suspected. Flagella occurred as before in
certain individuals only. No changes suggesting degeneration or breaking up
of the bodies were seen, and fission still seemed to be in progress. On this day
an unstained preparation was examined and a single large group of bodies
encountered, but no movement could be made out nor were flagella visible.
Preparations were also examined on the fifteenth and twenty-first days, but
the appearances were those seen on the twelfth day, except that groups were
more scanty. On the thirty-third day bodies were found with great difficulty,
and not only showed no further changes, but appeared to be showing signs
of dissolution. As the amount of blood had become very small, no further
examinations were made.
On the twelfth day a portion of this culture was removed in a sterile pipette
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and added to about double its volume of distilled water, and placed again in
the ice chest. A further portion was similarly added to normal salt solution.
Two days later prolonged search failed to reveal any structure resembling
the parasites.
On the fifteenth day about half the blood remaining was removed in a pipette
and added to another tube, the whole process being very carefully conducted to
avoid bacterial contamination. The tube was then enclosed in a moist chamber
and incubated at 35ºC. On the second day films were examined for the bodies.
Only a very few forms all showing marked degenerative changes, bordering on
dissolution, were seen. On the fourth day no trace of the parasites could be
observed. The preparation was, so far as could be seen under the microscope,
free from micro-organisms.
There could be no doubt from this case that, under the conditions described
by Captain Rogers, profound developmental changes in the bodies had taken
place. These appeared to be of the following nature and order of seluence:
enlargement together with changes in the large chromatin mass and the proto-
plasm, proceeding until the bodies are at least as large as a red blood corpuscle;
fission occurring at least twice and forming groups of two and four large oval
bodies loosely, or not at all attached ; formation of the vacuolic area and extrusion
of the cement substance ; flagellation of certain of the bodies, and the formation
of numerous fission forms many of which flagellate; finally, the formation of
large groups of closely aggregate forms of various sizes. The time taken in
this case for the different stages was as follows : large fully developed forms
appeared by the fourth day ; the vacuole, the extrusion of the cement substance,
and the early stages of the flagella formation, all first appeared on the fifth day.
Fully developed flagella were not seen till the sixth day. Elongate flagellate
forms were specially numerous on the sixth and seventh days. From the ninth
day onwards the formation of masses of pear-shaped and irregular forms was
the chief feature. Captain Rogers notes large forms on the second day and
flagellate forms on the third day. He does not note any further stages than
those seen on the fourth day, which correspond with those obtained by me on
the fifth and sixth days.
Case 2. —The patient had a greatly enlarged spleen and an irregular high
temperature. About .25 c.c. of blood was obtained by spleen puncture.
Bodies were rather scanty in a film taken at the time of puncture. In this speci-
men, after subsidence of the corpuscles, a slight clot formed in the plasma.
On the third day no development was noted. On the fifth day some large
forms not quite so large as in the fully developed bodies noted in Case 1 were
seen. Most of the bodies had undergone little or no change. On the tenth day