NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
Specimens for laboratory examination.
Appx. XXI.
(c)The exact nature of the examination required, and the reasons
rendering it necessary.
(d)In any case of suspected poisoning, a clinical summary. This
summary should state particularly the length of time which
elapsed between the consumption of the article and the
onset of symptoms, the symptoms observed in the order
of their occurrence, the present condition of the patient
or patients and the condition of other persons who have
partaken of the article or of similar articles from the same,
consignment.
These reports will be signed by the officer who actually took the
sample, and may be countersigned also by the officer at whose request
the sample was taken.
10. Reports on analyses carried out at the Military Food Laboratory
will be sent to the officer at whose request the sample was taken, a
copy being sent to the A. D. M. S. of the District concerned.
C.—PATHOLOGICAL MATERIAL FOR LABORATORY EXAMIN-
ATION.
SPECIMENS FROM SUSPECTED ENTERIC CASES.
1. Blood culture. —This is a most valuable method of diagnosing
cases. It is important to consider the date of onset of disease, and
not of admission to hospital only. Blood culture should also be carried
out in every relapse as soon as the temperature rises. Glassware
and the skins of patients in India are difficult to sterilise owing to
the presence of sporing organisms. A sterile syringe kept entirely
for blood culture work and for no other purpose, should be employed.
The skin should be thoroughly washed with soap and water over the
bend of the elbow, and the area painted with a solution made by
dissolving 0.5 per cent. each of brilliant green and crystal violet in
50 per cent. alcohol. Cover the area with sterile gauze and leave 10-15
minutes. This solution renders the skin quite sterile.
At least 3 c.c. of blood should be inoculated into the bile, and the
rubber cap then covered with melted paraffin.
2. Agglutination tests. —At the same time as blood is taken for culture
2 c. c. of the blood should be placed in a sterile tube and allowed to
clot firmly. Once the serum has separated it should be pipetted into
a Wright's capsule, and forwarded to the laboratory for Widal test.
Subsequent samples of serum should be forwarded to the laboratory
at intervals, e.g ., 10th, 14th, 18th, 22nd day of disease. It is to be
remembered that one single agglutination test is of no value in diag-
nosis in an inoculated individual. Tests on 26th and 30th day of disease
in addition to above tests may yield information of great value, and
should be carried out in suitable cases.
3. Collection of blood serum. —Two methods can be employed for
obtaining serum for agglutination tests, viz., collection in a Wright's
capsule or by means of vein puncture.
275
Appx. XXI.
Specimens for laboratory. examination.
As Dreyer's method of carrying out the agglutination test is in use
in all Military laboratories, collection by means of vein puncture is
practically always essential for the test to be properly carried out.
(a)Technique of collection in a Wright's blood capsule.—These
eapsules are obtainable on indent from Medical Store Depôts and in
emergency from the nearest military laboratory.
Their method of use is as follows :—
The dorsal aspect of the terminal phalanx of one of the fingers is
cleaned thoroughly with alcohol, and is pricked sufficiently deeply,
either with a bayonet pointed needle or glass pricker, to ensure a free
flow of blood. Before pricking the finger, or immediately afterwards,
a narrow bandage is wound tightly round it in such a manner as to
confine the blood in the distal phalanx.
Both ends of the capsule are now broken off. Holding the capsule
almost horizontally and below the level of the finger, the tip of the re-
curved limb is applied to the drop of blood. This now runs in by
capillary attraction and by siphonage, the air escaping through the
straight limb of the capsule. Enough blood will have been obtained
when the capsule is two-thirds full.
If, during the filling of the capsule, the blood ceases to flow freely
the bandage should be removed from the finger and re-applied.
The capsule is sealed in the following manner :—A small steady
flame is employed, either the by-pass of a Bunsen burner, or if this is
not available the flame of a spirit lamp held upright. The capsule
is first slightly warmed at the junction of the body with the straight
limb, and the distal end of this tube is sealed off by holding it in the
flame. It is now allowed to cool, and in so doing the imprisoned
rarefied air contracts and the blood is drawn into the body of the
capsule, leaving the orifice of the re-curved limb free. The orifice of
this re-curved limb is now sealed up in the flame.
The greatest care must be taken in all these operations to prevent
charring or heating the blood.
The capsule is labelled with the patient's name and date of collection
of blood, and is packed carefully for forwarding.
(b)Technique of collection by means of vein puncture.—The blood
is obtained from the forearm by means of aseptic puncture of one of
the veins which have been rendered prominent by getting the patient
to clench the fist and grasp the upper arm with the other hand, or by
applying a bandage round the upper arm. A fairly large-bore hypo-
dermic needle should be employed, and at least 3 c.c. of blood with-
drawn into the syringe. This blood is now transferred to a small
test tube to allow clotting and separation of the serum. The latter
is pipetted off from the clot, sealed in a small tube and labelled with
the patient's name and date of sample.
4. Fœces should be sent daily to the laboratory from the 8th day
of disease onwards. Specimens taken between the 10th and 21st day
of the disease are most likely to yield positive results. All specimens
should reach the laboratory within two hours after passage. When
276
277
K
Specimens for laboratory examination.
Appx. XXI.
this is impossible, as in the case of outstations, a small portion of a loose
stool should be emulsified, directly after passage, in 30 per cent.
" neutral" glycerin in 0.6 per cent. saline solution, and sent by post.
It is important that the glycerin should not be acid. Constipated
stools are usually worthless for bacteriological examination ; therefore
when the condition of the patient permits, the stool resulting after
saline treatment should be forwarded. Proper glass containers
must invariably be used.
5. Urine.—Specimens of urine should also be sent daily to the
laboratory after the 8th day from onset of the disease. Specimens
should be collected from the first urine passed after waking in
the morning. The first portion should be passed into a separate
receptacle, and about 20 c.c. of the middle portion of the urine
collected into a sterile bottle, and forwarded at once to the
laboratory. Urine from outstations is usually so contaminated
by the time the specimen reaches the laboratory that it is useless
for bacteriological examination. This may be overcome by add-
ing 0.8 c.c. of a 1-10,000 solution of brilliant green to 20 c.c. of
urine (to prepare 1-10,000 brilliant green solution make up a 1-10
solution in distilled water, and add 0.1 c.c. of this solution to 10 c.c.
distilled water). The urine with added brilliant green solution should
be despatched to the laboratory as quickly as possible. Catheter
specimens should be taken when required, but routine daily cathe-
terisation is for obvious reasons undesirable.
Very careful sealing and packing of receptacles containing blood
fæces or urine are required when they have to be transmitted through
the post. Effective sealing of bottles or tins may be accomplished with
melted paraffin wax or sealing wax. Straw, bran, or some similar
material, never paper, should be used for packing.
SPECIMENS FROM CASES OF SUSPECTED DYSENTERY.
6. When there is a laboratory in the station send the entire stool
whenever possible and note that the specimen should be sent as early
in the disease and as soon after passage as possible. The longer the
delay the less chance of a positive diagnosis.
7. Bacillary dysentery.—Dysentery bacilli die out rapidly in the
stool after passage and are only isolated as a rule in the early stages
of the disease i.e., during the first three days. Every endeavour
should therefore be made to ensure that the specimen is sent to the
laboratory directly the patient comes under observation and without
any delay after passage. When the specimen cannot reach the labora-
tory within two hours of being passed, a portion of the mucus should
be placed as soon as possible in 30 per cent. neutral-glycerin in 0.6
per cent. saline and forwarded to the laboratory. It is mast important
that neutral glycerin only should be used. The glycerin solution should
be tested with litmus paper from time to time and neutralised
if necessary.
A portion of mucus spread on a glass slide and allowed to dry
should also be forwarded from every case for a laboratory examination
Appx. XXI.
Specimens for laboratory examination.
of the type of cellular exudate. No pressure should be used for spread
-ing this film or the cells will be broken down. A useful method is to
place some mucus on a piece of litmus paper used for testing the reaction
of the stool and draw the mucus along the slide. The reaction of the
mucus and the time and date on which the stool was passed should
be invariably entered on the laboratory report.
Instructions should be given to subordinates that in cases suspected
of suffering from dysentery a clean dry bedpan free of antiseptics
should be used and also that patients should first pass their urine into
a separate receptacle in order that no urine should be mixed with the
stool in the bed pan.
8. Amoebic dysentery—E. histolytica begins to degenerate directly
it leaves the intestine. Movement ceases as a rule after one hour and
diagnosis frequently becomes impossible. In cases in which amoebic
dysentery is suspected, but in which it is impossible for the specimen
to reach the laboratory within at the latest two hours after being
passed, mucus (preferably blood stained) should be spread on three
coverslips. Before the films dry the coverslips should be dropped
film surface down into Schaudinn's solution and left for fifteen
minutes. They should then be transferred to a small bottle con-
taining 70 per cent. alcohol (with a little cotton wool at the bottom
of the bottle to avoid breakage in transit) and be forwarded to the
laboratory.
Schaudinn's solution.
Saturated solution of mercuric chloride in
normal saline . . .
2 parts
Absolute alcohol . . .
1 part
Glacial acetic Acid . . .
3-5 %
In every case a dried film of mucus prepared as described above in
para. 7 should be forwarded for examination of the cellular exudate.
Fæces for examination, for cysts should be selected not from loose
stools but from solid fæces, which should be thoroughly emulsified
in Weigert's iodine solution and forwarded to the laboratory.
Weigert's iodine solution.
Iodine . . .
1 gramme.
Potassium iodide . . .
2 grammes.
Water . . .
100 c.c.
N.B. —Cysts are not found in cases of acute dysentery.
9. Stools should be forwarded daily to the laboratory until a
positive result is obtained or until information has been received from
the D. A. D. P. that further specimens are not required.
10. In every case of dysentery medical officers should take the
opportunity of examining specimens of mucus in the clinical side
278
Specimens for laboratory examination.
Appx. XXI,
room directly after the specimen has been passed. Small portions of
mucus only, spread thin by the pressure of the coverslip, should be
examined. Only motile amoebae containing red blood cells should be
diagnosed as E. histolytica .
SPECIMENS FROM CASES OF DIARRHŒA.
11. Acute diarrhoea may be caused by organisms of the food-
poisoning group or by organisms of the dysentery group. Fæces from
such cases should therefore be forwarded to the laboratory as described
above for suspected enteric cases. In all cases where vomiting has been
present vomited material should be sent in a similar manner. In food
poisoning cases the suspected food should invariably be forwarded at
the same time if possible, packed in ice.
SPECIMENS FOR EXAMINATION OF FÆCES FOR OVA OR WORMS.
12. A saline purge should be given overnight and a small portion of
the stool forwarded to the laboratory after being thoroughly emulsifi-
- e d i n t h e f o l l o w i n g s o l u t i o n : —
Acid carbolic. . .
1 part
Glycerin . . .
2 parts
Lactic acid . . .
1 part
Water . . .
1 part
SPECIMENS OF FAECES FOR ROUTINE EXAMINATION FOR CARRIERS OF
ENTERIC BACILLI, DYSENTERY BACILLI, OR E. HISTOLYTICA.
13. Enteric group.—Constipated stools are worthless for bacterio--
logical examination, therefore only loose stools resulting from a saline
purge should be forwarded. Specimens should be forwarded in the
glycerin and saline solution as described in para. 7 above, in those cases
in which the specimen cannot reach the laboratory within two hours.
Three such examinations should be carried out as a routine before
a negative result is accepted.
14. Dysentery group :
—
(a) Bacillary dysentery.—Bacteriological examinations for dysen-
-tery bacilli, will also be carried out in the laboratory on
the specimens referred to under para. 13. Enteric group
above. It should be noted that such examinations are rarely
positive in the absence of mucus, and therefore the pre--
sence of mucus should be looked for in these specimens, and
a sample if present forwarded separately to the laboratory.
( b ) A m o e b i c d y s e n t e r y . — A sa
m p l e o f f æ c e s e m u l s i f i e d i n W e i g e r t ' s
iodine solution should be forwarded to the laboratory
on three successive days A saline purge should not be
administered, as formed stools are required for cyst exa-
279
k 2
1
Appx. XXI.
Specimens for laboratory examination.
minations. These specimens should also be macroscopically
examined for presence of mucus. Routine examinations
will thus be as follows : —
Three for enteric group and for dysentery bacilli.
Three for E. histolytica cysts.
Macroscopic examinations for mucus should be made on
these six specimens.
TISSUES FOR HISTOLOGICAL EXAMINATION.
15.Portions of tissues, new growths, etc., forwarded for histolo-
gical examination should be dealt with as follows :
—
The tissue should be obtained as fresh as possible, and if for sending
by post, cut into cubes from 2 to 4 millimetres in thickness and
placed, without previous washing with water, in a glass stoppered
bottle containing 10 per cent. formalin in normal saline solution.
Particulars as to the site of new growth and brief clinical details
should be forwarded with the specimen.
If a properly equipped laboratory is available, the whole growth
or tissue should be handed without delay to the pathologist:
METHOD OF DEALING WITH THE BRAIN OF A DOG SUSPECTED OF
RABIES.
16.Method of removing the brain of a suspected rabid animal for
laboratory examination.—The first procedure is to open the skull and
expose the brain.—Wash the head of the dog with an antiseptic,
hammer
as carbolic acid, phenyl, etc., then take a ammer and with a few
sharp blows fracture into many pieces through the intact skin, the
top and sides of the brain cavity. With a knife throw back the skin
and remove very carefully the pieces of fractured bone, in this way
exposing the brain. Incise the membranes covering the brain and
divided the brain equally down the centre into two longitudinal halves.
If the skull vault is well broken, each half of the brain can be lifted
up with the aid of a knife and forceps.
NOTE.—In removing the brain of rabid animals the greatest care must be exercised
as the brain and saliva are infective, and, therefore, should never be allowed to come in
contact with the hands. It is best to wear a pair of old riding gloves when carrying out
this operation.
17.Preservation of the brain.—For the microscopical method, a
special portion of the brain is required, viz., the hippocampus major.
To those who are unacquainted with the anatomy of the brain, it is
best to remove the brain in two longitudinal halves as directed above.
If the brain is a large one, e. g., horse, cow, great dane, etc., and therefore
difficult to preserve only send one hall, as there is a hippocampus
major on each side. The brain is lifted up between the forceps and
knife and placed in a large wide mouthed bottle, with a capacity of
at least two pints, a layer of cotton Tool being placed first at the bottom
of the bottle.
280
Specimens for laboratory examination. Appx. XXI.
The bottle is now filled up to the brim with one of the following
solutions :
—
(A) Zenker's fluid :—
d r a c h m s
Pot. Bichromate . . .
5
Mercury Perchloride . .
7
Glacial Acetic Acid . . .
7
(or ordinary Acetic Acid) . . .
21
Water . . . up to 20
ounces.
(B) Rectified spirit.
(C) Methylated spirit.
Very inferior results are obtained with Methylated Spirit. It is
therefore recommended that one of the first two fluids be used wherever
possible. Large quantities of preservative fluid are always required.
18. Packing and despatch.—A layer of cotton wool is placed at the
bottom of the bottle and the bottle is filled up to the top with the
preservative fluid. This prevents jarring during a journey and con
- s e q u e n t l y t h e b r a i n i s n o t b r o k e n u p a n d e m u l s i f i e d . I n n o c a s e
should the brain be wrapped round with cotton wool as it prevents the
penetration of the preserving fluid. The lid of the jar is now sealed
and the jar is packed in a box carefully with saw dust, etc.
A descriptive account of the illness of the animal should always
accompany the brain, and also the full address of the person to whom
the results of the examination are to be sent.
Medical and veterinary officers should also send a small frag
- m e n t o f t h e b r a i n i n 5 0 p e r c e n t . g l y c e r i n a n d d i s t i l l e d w a t e r i n c a s e s
of animals which they regard as undoubtedly suffering from rabies,
as " Street Virus " is always required at Pasteur Institutes.
SPUTUM FOR BACTERIOLOGICAL EXAMINATION.
19. It is advisable to obtain the first sputum that is brought up in
the early morning.
Particular care should be taken when collecting bronchial secretion,
that the sputum is coughed up and is not simply obtained from the
upper air passages or mouth.
It is convenient, in forwarding the specimen, to use a stoppered
sputum bottle.
These bottles can readily be sterilized before use by rinsing with
a few drops of absolute alcohol or methylated spirit which is then set
alight.
MATERIAL FOR AUTOGENOUS VACCINES.
20. Autogenous vaccines, for cases of furunculosis, acne, etc., can
be prepared, provided a specimen of pus, collected with aseptic pre
- c a u t i o n s , b e f o r w a r d e d .
281
Appx. XXI.
Specimens for laboratory examination.
An unbroken pustule should be selected and the surface sterilized
by drubbing with cotton wool moistened in absolute alcohol.
The pus is obtained by plunging the point of a sterile capillary
pipette into the centre of the pustule and gently aspirating a portion
of the contents into the capillary stem by means of the teat fitted to
the end of the pipette. The end is then sealed off in the flame.
BLOOD FILMS FOR MALARIA PARASITES, LEISHMANIA, BLOOD
DISEASES. ETC.
21. The ordinary method.—The blood should be spread on a clean
microscope slide in as thin and even a film as possible. This is best
attained in the following manner :—A small drop of blood, obtained
by pricking the dorsal aspect of the terminal phalanx of the finger,
is touched lightly with the end of the slide. The slide, with the drop
of blood thus transferred, is placed on a horizontal surface and with
the edge of a second slide, which is held at an angle of 45 degrees, the
drop is distributed in an even film over the surface. A film with its
edges parallel to the edges of the slide can be obtained by using as a
spreader a slide broken so as to give a narrow end. Blood films should
be protected from flies and dust. The film may be stained by Leish-
man's, Giemsa's or other appropriate stain.
22. The thick film method.—There are several varieties of this
method, one of the best being as follows :
—
Four large drops of blood are placed at the corners of a small
square, ½ i n. by ½ i n. , near t he cent re of t he sl i de. Wi t h
a round needle or glass rod they are then pooled so that the
blood covers the ½ in. square thickly and evenly : " pud-
dling " must be avoided.
The slides are now laid flat on the table, are covered with a
Petri dish and are allowed to dry completely. This is the
most important point of the whole process. A thick film- may
appear to be dry in half an hour, but the leucocytes have
not yet emigrated to and become adherent to the slide.
At least two hours at room temperature, or an hour in the
37°C. incubator, is required : otherwise the film gets washed
away during subsequent manipulations.
Lay the perfectly dry thick film,—surface upwards,—flat on
a 'staining rack. Flood the slide very gently with the
following mixed solution :
—
Glacial acetic acid : 25 per cent. in
distilled water
.
. 4 parts
Tartaric acid crystalline : 2 per cent. in
distilled water
.
. 1 part
This solution dehaemoglobinizes the film, and the process should
be watched. An ordinary thick film will he completely
dehaemoglobinized in 5 to 10 minutes, but films with
thicker patches may require a little longer.
282
Specimens for laboratory examination.
Appx.
As soon as dehaemoglobinization is complete, drain off the
fluid by tilting the slide. Next, flood the slide with methyl
alcohol. Allow to remain on for five minutes. The film
is now fixed.
Drain off the methyl alcohol and wash the film very thoroughly
in distilled water. Every trace of acid must be removed
from the film, or the subsequent staining will be unsatis-
factory.
Stain the film with Giemsa's stain or Leishman's stain, one
drop to one c.c. of distilled water, for 10 minutes. Differ-
entiate in distilled water. Do not blot the film, but allow
it to dry in air, placing the slide tilted against any vertical
surface, with the film side downwards to protect it from
dust.
Examine with the 1-12th-in. oil immersion lens and a fairly
high, eg., No. 6 ocular. The leucocytes are seen to be
evenly scattered, field by field, over the half-inch square,
which can be rapidly examined for malaria parasites,
L. donovani, etc. As contrasted with control thick films
for healthy blood, the leucopenia of kala-azar at once
becomes most strikingly apparent.
Pus FILMS FOR MICROSCOPICAL EXAMINATION.
23. These films should be prepared in the same manner as their
blood films(see para. 21).
In preparing films for examination for the gonococcus, the sample
of pus should be collected on the patient's rising in the morning and
before he has urinated.
D. RULES FOR PRESERVING PACKING, AND DESPATCHING
SPECIMENS TO CHEMICAL EXAMINERS FOR ANALYSIS.
1. The suspected viscus or other materials to be sent for examination
should be enclosed in a glass bottle or jar, fitted with a stopper or sound
cork.
2. If the material sent is liable to decomposition, it should invari-
ably be preserved by one of the following methods :—
(i) In cases of suspected poisoning in man, other than alcoholic
poisoning, the material sent should be immersed in spirit
of wine. The spirit should be sufficient in quantity to
cover the material immersed in whatever position the vessel
containing it may be held, and should not bear a less
proportion to the bulk of such material than one-third.
Care should be taken that common bazar spirit is not used.
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