NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
( 12 )
Films prepared in this way should be allowed to dry in the air and then
fixed by immersion in absolute alcohol for ten minutes.
Staining the
specimen.
It is better for the beginner to get accustomed to one stain than to
attempt many different methods+, and the best stain for our present purposes
is that recommended by Romanowsky. This is a triple stain consisting of a
combination of a particular kind of eosin and medicinal methylene blue. A
modification of it called Leishman's stain can now be obtained in the form
of compressed tabloids * which are very convenient to use. Each tabloid
should be dissolved in 10 cubic centimetres of pure methyl alcohol, and the
stain is then ready. If this stain is used, previous fixation of the blood film is
unnecessary. After the film has dried in the air it is covered with a few drops
of the stain. A few seconds afterwards, and while the stain is still liquid, an
equal quantity of distilled water is added to the stain, and the slide is moved
about so as thoroughly to mix the water with the stain. This mixture is
allowed to remain on the film for from ten to twenty minutes, and is then
washed off with a stream of distilled water. The slide is then allowed to
dry in the air. It is not necessary to cover the film with a cover glass ;
the oil for the oil-immersion lens may be placed directly on the film of
blood.
When it is desired to send specimens of blood for examination in a
laboratory dry films taken in the manner just described must be sent, but
they need not be fixed or stained ; each slide should be wrapped in a piece of
clean paper on which the particulars of the case are written.
Microscopic
examination.
When the making and preparing of blood specimens has been sufficiently
practised one will take up the task of microscopic examination. For this
purpose a good microscope provided with a one-twelfth inch oil-immersion
objective is necessary. We must commence our examinations not with
specimens of blood from patients in hospital but with specimens of ordinary
normal blood because " It is impossible to make reliable examinations of the
blood for malarial parasites without first being familiar with the ordinary
appearances of normal blood and the more common pathological changes.†"
* From Messrs. Burroughs, Wellcome & Co., London, and from many chemists in
India,
† Lectures on the malarial fevers, by W. S. Thayer, M.D., Professor of Medicine in
the Johns Hopkins University.
( 13 )
The. necessity
of being
familiar with
the appear-
once of
normal blood.
This axiom cannot be too strongly impressed upon all who enter upon
the microscopic study of malaria parasites, and many pages might be filled
with an account of errors that have been made by observers who have
confused normal constituents of the blood, such as blood platelets and even
leucocytes, to say nothing of such common pathological changes in the red
corpuscles as vacuoles, crenations, fissures, etc., with malaria parasites.
Before commencing to study the appearances of malarial blood it is essen-
tial, therefore, for every one to make himself familiar with the appearances
of normal blood, and to be able to distinguish differences in the colour, size,
and shape of the red corpuscles, and to recognise such changes in them as
crenations, vacuoles, and fissures, and also to be thoroughly familiar with
the different varieties of leucocytes and with the blood platelets.
A description of these constituents of normal blood cannot be included in
this book, and it must suffice to repeat yet once again that learning to recog-
nise these constituents and the various pathological changes just mentioned
both in preparations of fresh blood and in stained preparations, must be the
first task which the beginner will set himself seriously to undertake, and
that he will not consider his task complete until he is sure that when he
comes to examine specimens of blood from patients who have fever he will
not mistake for malarial parasites any of the appearances (including particles
of dirt, deposits of stain, etc.) with which he has become familiar during the
examination of normal blood. Having attained a good knowledge of the
appearances in specimens of normal blood the student may commence to
search for malaria parasites, and at the outset it is necessary to mention
that the reasons why beginners make so many mistakes in regard to the find-
ing of malaria parasites are firstly because they are not familiar with the
appearances of normal blood and secondly because they do not search in
suitable cases at the beginning of their study. The majority of men who
are commencing the subject expect to find parasites in the most unlikely
cases—cases the temperature charts of which show no sign of intermittency
or cases of chronic cachexia which have been drenched with quinine. Even
an experienced observer would not be able, in all probablity, to find parasites
in such cases. The truth is that a hospital is not, as a rule, the place in
which one should begin the search for malaria parasites. The following
( 14 )
How to obtain
specimens of
malarial
blood.
plan is much more likely to be attended with success. Find a native village
in which a large proportion of the young children are suffering from enlarge-
ment of the spleen, and in the evening take the temperatures of half a dozen
children whose spleens are enlarged. It will almost certainly be found that
some of the children have a rise of temperature although they may be
playing about apparently quite well. In return for a small present these
children will allow their blood to be examined as frequently as may be
desired, and the finding of malaria parasites in their blood is generally an
easy matter. When a child whose blood contains malaria parasites has
been found in this way the course of the temperature and the changes
which the parasites undergo from day to day can be studied with great
ease.
As regards the actual examination of the film the beginner should
remember above all that—
(1) the parasite must be looked for inside a red blood corpuscle—
so-called "free " parasites are almost never seen ; and
(2) the malaria parasite has a very definite structure and appearance,
and if once seen cannot well be mistaken for anything
else; nothing which is at all "indefinite" is likely to be a
parasite.
How to
examine the
film.
Preparations of fresh blood should be examined first with a one-sixth
inch objective ; if the preparation is a successful one, the red blood corpuscles
in many parts of the specimen will be found to be lying flat in a single layer
with their edges nearly touching each other but not overlapping, and these
are the parts of the specimen to be examined for parasites. Nearer the edge
of the cover glass the corpuscles will be found heaped up and overlapping
each other. Having found a suitable field, a small drop of cedar wood
oil is placed on the cover glass and the oil-immersion lens turned on. A
substage condenser and a low-power eyepiece must be used, and the iris
diaphragm should be nearly closed so that only a moderate amount of light
passes through the specimen ; if the illumination is too brilliant the parasites
may be easily missed.
A beginner should not attempt to find small unpigmented parasites ; he
should search for large pigmented forms.
( 15 )
How to find
the parasite.
On searching each corpuscle he will presently come across one
in which fine black dots of pigment are seen. On focussing carefully
he will find that these dots are contained in a transparent mass
of pale protoplasm which is undergoing amoeboid movement inside
the corpuscle. After a short time the movements will become less
marked, and the outline of the body will become more distinct. Careful
watching will convince the observer that he is looking at a living
parasite. Once having found a parasite, the beginner will know what sort
of body to look for, and will easily be able to educate himself to pick out
the smaller forms.
Stained films.
Stained films can be examined at once with the one-twelfth inch objective,
and the illumination should be as brilliant as possible. The drop of cedar
wood oil should be placed on one of the side edges of the film, and different
fields along this edge should be searched until the examination is ended. By
the method of staining which has been described here the red blood corpuscles
are stained pink and the blood platelets and the nuclei of the leucocytes a
deep ruby-red.
Leucocytes.
Polynuclear leucocytes will be recognised by their large irregular
nucleus ; their body protoplasm is unstained and contains fine red
granules.
Large mononuclear leucocytes have generally a large oval or horse-shoe
shaped nucleus and faintly blue stained body protoplasm which is free from
granules. They may contain grains of intensely brown or black malarial
pigment (melanin) which should be carefully looked for as they are a sign of
recent infection.
Lymphocytes have a round, ruby-red nucleus surrounded by a thin rim
of blue stained protoplasm.
Eosinophile leucocytes have generally two pear-shaped nuclei joined
together at the top, and numerous large, round, red granules scattered
through the cell, which make them very easily recognisable.
Blood
platelets.
The blood platelets are deep ruby-red, and will generally be found in
groups looking something like bunches of grapes. Many beginners mistake
them for the "spores" of malaria parasites.
Malaria parasites are very easy to detect in the red blood corpuscles by
( 16 )
this method of staining. Young "ring " forms of the parasite in the red
blood corpuscles present the following characters :—
The parasite.
(1) A brilliant red dot at the periphery (the nucleolus of the
parasite).
(2) A clear space almost unstained (the nucleus of the parasite).
(3) A circle of blue stained protoplasm surrounding the clear nucleus
(the body of the parasite).
Larger forms of the parasite present the same characters, viz.:—
(1) One or more red patches of chromatin.
(2) A clear space.
(3) A large blue stained body, in which grains of brown or black
pigment can be plainly seen.
Changes in the
red corpuscles.
The red corpuscles containing parasites may be variously altered. In
Benign Tertian infections, for example, the corpuscles containing parasites
may be swollen to more than double their original size, and with this stain
they may be infiltrated with red stained dots to such an extent that the
parasite itself is almost hidden. When corpuscles studded with these red
dots (Schiiffner's dots) are seen, we may be sure that the case is one of an
infection with Benign Tertian parasites. When the parasites have arrived at
the segmenting stage it will be seen that the patches of chromatin have
divided up into a number of parts, each of which represents a "spore."
A thin rim of corpuscle may still be seen round the group of red stained
spores, and near the centre of the group a mass of black pigment will be
collected.
The sexual forms of Benign Tertian and Quartan fevers are difficult to
distinguish in stained specimens from the full grown asexual forms, but the
sexual forms of Malignant Tertian fever, viz., " crescents," will be very easily
recognised from their characteristic shape and from the grains of pigment
collected at their centre. They are stained light blue with Romanowsky's
stain, and near their centre, partly hidden by the pigment, a mass of red
stained chromatin will be seen.
We may take the opportunity here to explain the terms Benign
Tertian, Quartan, and Malignant Tertian which have just been mentioned.
( 17 )
Clinically the malarial fevers can be separated into three chief types,
namely :—
(1) A comparatively mild type in which (counting the day of a febrile
paroxysm as the first day) the febrile paroxysms recur with
great regularity every third day, the intervals * between
the paroxysms being of 48 hours' duration (Benign Tertian
fever).
(2) A severe type in which the paroxysms also recur approximately
every third day but not with such regularity as in the first type
and in which " pernicious " symptoms are very liable to develop
(Malignant Tertian or æstivo-autumnal fever).
(3) A comparatively mild type in which (counting the day of a
febrile paroxysm as the first day) the febrile paroxysms
recur with great regularity every fourth day, the intervals
between the paroxysms being of 72 hours' duration (Quartan
fever).
The three
species of
parasite,
Their
differences.
It has been found that these different clinical types are due to infections
with different species of the malaria parasite ; that there are, in fact, at least
three distinct species of the malaria parasite, each of which causes a type of
fever different from that which is caused by the other species. These species
are called respectively the Benign Tertian parasite, the Malignant Tertian
parasite, and the Quartan parasite.† It is important to be able to distinguish
these species from one another by a microscopical examination of the blood.
In the very young stage before the parasites have acquired pigment, this is a
difficult matter, but in later stages it is easy. At a microscopical examina-
tion the points of distinction depend on the following observations :—(I)
the stages of the parasite which are found in the peripheral (finger) blood; (2)
the effect of the parasites on the red blood corpuscles containing them; (3)
the character of the amoeboid movements of the parasite ; (4) the number
of spores into which the parasite divides up ; (5) the character of the sexual
forms ; (6) the character of the pigment seen in the parasite.
*See the definition of interval on page 1. Do not confuse it with intermission.
† The English names only are given because as regards Latin names no two authori-
ties employ the same terminology.
0
( 18 )
The differences between the three species of parasite in the above
respects may be tabulated thus :—
Species of
parasite.
Quartan
Benign
Tertian
Malignant
Tertian.
Stage found
in the
peripheral
(finger) blood.
All stages
All stages
Only young
''ring"' forms
and crescents.
Very rarely
an occasional
segment ing
form.
Effect of the
parasites on
the red blood
corpuscles
containing
them.
The corpuscle
is practically
unaltered.
The corpuscle
is very much
swollen and
decolourised.
The corpuscle
is generally
shrunken and
decolourised.
Never swol-
len.
Character of
the amoeboid
movements of
the parasite.
Very sluggish
so that the
parasite is
generally
oval or more
or less rect-
angular.
Very
active
so that the
parasite is
irregular in
shape.
Very active and
with
a great
tendency
to
assume
the ring
form.
Number and
arrangement
of spores.
8-10.
They
occupy the
whole of the
corpusele
and are
arranged
with great
regu1arity
" like
a
daisy."
20-23. Not so
re g u 1 a r 1 y
arranged.
9-10.
T h e
segmenting
form occupies
only about
1/2 — 2/3 of the
red corpus-
cle.
Charac-
ter of the
sexual
forms.
Round.
Rarely
seen.
Round.
Crescent-
shaped.
Charac-
ter of the
pigment.
Coarse
black
grains.
Fine
brown
granu-
les or
rods.
Fine
brown
granu-
les or
rods.
Hints.
From the point of view of diagnosis by a microscopical examination the
following hints may be useful.
If in a film we find a " crescent," we know at once that we are dealing
with a case of Malignant Tertian fever.
If, at different examination of the same case, we find nothing but small
ring forms, the case is also probably one of Malignant Tertian. Small rings
and crescents are characteristic of Malignant Tertian infections.
If we find a large irregularly shaped parasite in a corpuscle which is
distinctly larger than the surrounding ones, we may be sure that this is a
Benign Tertian parasite.
If we find large regularly oval or rectangular parasites in corpuscles which
are practically unaltered in size and colour, these are probably Quartan parasites.
( 19 )
Search for segmenting forms should be made an hour or so before the
time of onset of the febrile paroxysm. If one can secure a case of regularly
intermittent Quartan fever, very perfect segmenting " daisy like " forms will
be easily found in the peripheral blood.
2.—The examination of specimens of blood for other parasites.
The spirillum
0bermeyeri.
The Filaria
nocturna.
Trypanoso
mala.
The possibility that other parasites than those of malaria may be present
in a blood film must always be kept in mind during the examination of the film,
and occasionally we should turn our attention away from the careful scrutiny
of the red blood corpuscles and concentrate it upon the clear spaces between
the corpuscles, focussing upwards and downwards with the fine adjustment
especially with a view to ascertain the presence or absence of the spirillar or
spirochete organism which is the cause of relapsing fever. (Plate II, g.) This
organism is about three times as long as a red blood corpuscle, but it is exceed
ingly thin and therefore is not easily detected in stained preparations unless the
observer's attention is concentrated upon the attempt. In the examination for this
parasite as well as for other parasites which occur free in the blood plasma, such
as trypanosomes and embryos of filariæ, it is best to employ in the first place
specimens of fresh blood. The fresh film to be examined for the spirochetes of
relapsing fever should be taken during a period of pyrexia and examined with
a one-sixth inch objective. The movements of the parasites cause a vibratory
disturbance of the corpuscles which plainly indicates that a living organism
is moving about amongst them. Afterwards dry films should be prepared
and stained and examined with the one-twelfth inch objective. The fresh
film to be examined for filarial embryos should be taken as late in the even-
ing as possible, because as a rule they appear in the peripheral blood at night-
time only. The examination should be made with a two-thirds inch objective
with which, owing to their comparatively large size, the embryos can be plainly
seen violently pushing the corpuscles about in all directions. (Plate II, d.)
Trypanosomes (Plate II, f) have not been found in human blood in India
up to the present but, they have been found in human blood in Africa where
they are the cause of the prolonged and fatal fever called trypanosomal fever
and of the deadly disease called sleeping sickness. They occur, however,
in many animals in India and one species, Trypanosoma lewisi, can be easily
studied in the blood of rats. Another species, Trypanosoma evansi, is the
cause of the fatal disease of horses, oxen, mules, camels, etc., named surra,
c 2
( 20 )
The Leish-
man-Donovan
parasite.
There is still one more parasite to be thought of during our examinations
of human blood, but as it is very rarely present in the peripheral blood and
then is present in the leucocytes only, it is not likely to be encountered
during the examination of films taken for the purpose of diagnosing malaria.
It is known as the Leishman-Donovan parasite and is the cause of the
fatal disease called kala azar which occurs in Assam, the Madras Presidency
and some other parts of India. In cases of kala azar the spleen and
nearly always the liver are greatly enlarged, and in order to find the parasite
it is necessary, as a rule, to obtain blood from one or other of these organs.
The operation of puncturing the spleen in this disease is by no means free
from danger and should not be undertaken if it is possible (as it usually is)
to arrive at a correct diagnosis by any other means. It is far preferable
from the point of view of the safety of the patient, as Sir Patrick Manson
has suggested, to puncture the liver than the spleen, and although the
parasites sometimes are not present in the liver in such large numbers as
in the spleen, they are usually sufficiently numerous for a diagnosis to be
made with ease. If one has to puncture the spleen the precautions which
must be taken are : (1) do not use a needle with larger calibre than that of
an ordinary hypodermic needle ; (2) sterilize the needle and the skin of the
patient ; (3) with the left hand press in the abdominal wall so that it is in
close contact with the spleen while the puncture is being made ; (4) make
the stab firmly and to the full length of the needle so as to ensure that the
needle enters the spleen immediately it has passed through the abdominal
wall ; this is very important because if the needle passes through the
abdominal wall but fails to enter the spleen, at the first stab its point will
scratch the surface of the spleen and cause bleeding ; (5) when the needle is
in the spleen do not move it about with the object of obtaining bits of
" splenic pulp " ; the blood which immediately wells up in the syringe on
gently withdrawing the piston will contain numerous parasites if the case is
one of kala azar ; (6) after the operation compress the point of puncture
with a pad and firm abdominal binder and make the patient rest in bed in
the recumbent posture for 24 hours. The blood obtained in this manner
should be spread in thin films on glass slides, dried in the air, and fixed and
stained in the same manner as for malaria parasites. The examination is to
be made with a one-twelfth inch objective, and the parasites will be recognised
PLATE II.
Line Block.
Survey of India Offices, Calcutta, 1908.
a, Egg of Ascaris lumbricoides; b, egg of Trichocephalus trichiurus;
c, egg of Ankylostoma duodenale; d, embryo of Filaria bancrofti in
blood; e, method of taking blood for the serum test; f, a trypanosome
in blood; g, three red blood corpuscles (stained) and four spirilla or
spirochetes; h, four Leishman-Donovan parasites as they appear when
free (stained); i, a leucocyte containing many Leishman-Donovan parasites
(splenic blood, stained).
( 21 )
very easily as small oval or round bodies with a very definite contour
lying in large numbers inside the leucocytes and free in the plasma. (Plate
II, h and i.) Each has two masses of chromatin, one being large and round
or oval, the other small and rod shaped.
3.—The method of collecting samples of blood for serum diagnosis and for
the culture of bacilli.
Every practitioner in the tropics should know how to examine for and
how to recognise the parasites dealt with above, but it is not within his
province to undertake bacteriological methods of diagnosis. It is sufficient
if he knows how to collect and send suitable material to a laboratory where
these methods can be carried out in a trustworthy manner.
How to collect
blood for the
scrum test.
To collect blood for examination by the serum test (for the diagnosis of
enteric and Malta fevers) one requires (1) small glass capsules of the shape
shown in the figure (Plate II, e); these can be obtained from any laboratory,
and a supply of them should always be kept, (2) a spirit lamp, (3) an unused sur-
gical needle, (4) alcohol or ether for sterilizing the skin, (5) a pair of forceps.
To collect the blood proceed thus : (1) place all the required articles on
a sheet of clean glass or paper on a table by the bedside of the patient ; (2)
light the spirit lamp ; (3) sterilize the tips of the forceps by passing them
through the flame and the two closed ends of the blood capsule by the same
method ; (4) with the forceps break off the tips of the glass capsule so as to
open both ends of it and lay it down on the clean glass or paper on the
table ; (5) sterilize the tip of the patient's finger with alcohol and dry it
thoroughly with a sterilized cloth ; (6) let the patient's hand hang down-
wards so that the blood collects in it and wind a corner of the sterilized
towel tightly round the base of the finger so that the tip of the finger
becomes congested with blood ; (7) prick the finger tip moderately deeply,
and a large drop of blood will immediately flow ; hold the open end of the
curved limb of the capsule in the blood, the bulb of the capsule being at a
lower level (see Plate II, e) :. (8) when the capsule is half full of blood remove
it and seal the open end of the straight limb by keeping it for a few seconds
in the flame ; (9) seal the open end of the curved limb by the same method ;
(10) place the capsule in cotton wool in a small box and despatch it with full
particulars of the case to a laboratory.